Application Of Electric Pulses Research Articles

Brain stimulation for surgical epilepsy

Direct brain stimulation is an emerging treatment of epilepsy especially in patients that are not candidates for epilepsy surgery. Several different approaches of brain stimulation in epilepsy have been developed: stimulation is applied to interrupt epileptic networks in subcortical structures or a stimulus is directly applied to the seizure onset zone. Scheduled stimulation targets mainly subcortical structures like the anterior thalamic nucleus or the centromedian nucleus of the thalamus. The anterior nucleus of the thalamus was studied in a randomized trial in humans. Several case series reported reduction of seizures targeting other subcortical structures. Scheduled stimulation of the seizure onset zone in the hippocampus has also been shown to be safe and effective in a small number of patients. The application of electrical pulses to test for certain brain functions has long been established for the purposes of brain mapping. Traditionally stimulation at 50Hz for several seconds has been used. This stimulus frequently causes afterdischarges or seizures. Afterdischarges can be terminated by applying a very brief stimulus at the same frequency. Responsive stimulation is based on detection on this principle. Seizures are recorded intracranially and a high-frequency pulse applied whenever seizures evolve electrically. An automated implanted device for seizures detection and stimulation has been developed and shown to be safe for human use. A large clinical trial is currently ongoing. In conclusion, the optimal target and mode of stimulation for the treatment of epilepsy remains under investigation and requires large and costly controlled trials.

Limb Sparing Treatment of Bleeding Melanoma Recurrence by Electrochemotherapy

Electrochemotherapy is an effective local treatment for tumors that combines administration of a chemotherapeutic drug with the subsequent application of electric pulses to the tumor. In addition, it was also found to have a vascular-disrupting effect. We report a case of limb-sparing treatment of bleeding melanoma recurrence by electrochemotherapy After intravenous application of bleomycin (15,000 IU/m2), 15 runs of electric pulses were applied by hexagonal needle electrodes (1.7 cm in diameter) in the center of the bleeding melanoma recurrence, and an additional 10 runs of electric pulses were delivered via plate electrodes (8 mm) on the rim of the tumor. Immediately after the administration of electric pulses, the bleeding stopped and did not recur. Crust formation was observed and the lesion decreased in size in a matter of weeks. We conclude that electrochemotherapy should be considered as a treatment option when dealing with bleeding melanoma recurrences as well as a limb-preserving treatment.

Double-Pulse Approach of Electrogenotherapy: An Analysis at the Single Cell Level

Plasmid gene transfer and expression can be obtained by the application of electric pulses to a mixture of cells and plasmids (electrogenotherapy, EGT), but clearly, the transfer to and across the nuclear envelope remains a problem. A biological approach showed that EGT was more effective during mitosis as expected. Schoenbach and Beebe showed that nanosecond pulses may affect the organelles (such as the nucleus). Therefore, we made an approach of the alteration of the nucleus induced by a short high electric pulse (musHV, up to 9 kV/cm, 5 mus) a few seconds after EGT (10times, 0.7 kV/cm, 5 ms) needed to introduce the plasmid in the cytoplasm. This was obtained by a digitized fluorescence approach at the single cell level, using propidium iodide as a probe with high affinity to nucleic acids. The first train of pulses (EGT) increased the mean fluorescence and size of the nucleus, with a fluorescence saturation level reached in less than 2 min. Mean fluorescence level and volume were maintained along the next 10 min. The application of a musHV pulse affects the mean fluorescence level and fluorescence repartition in the nucleus without additional modification of volume.

Resistance transition in metal oxides induced by electronic threshold switching

Metal oxides can display resistance transition by the application of suitable electrical pulses, and this behavior is exploited for the development of resistive-switching memory devices. Here we report time-resolved electrical measurements demonstrating that the transition from the insulating to the metallic-conductivity state in NiO is first triggered by a threshold switching, i.e., an electronic transition to a highly conductive state typically observed in disordered semiconductors. Evidence for the possibility of subnanosecond transitions to low resistance is shown. Numerical simulations of threshold switching and Joule heating are finally presented, highlighting the role of the parasitic capacitance in the set transition.

Comparison between hypoxic markers pimonidazole and glucose transporter 1 (Glut-1) in murine fibrosarcoma tumours after electrochemotherapy

Background. Tumour hypoxia occurs as a result of an inadequate supply of blood-borne oxygen due to the disorganized and chaotic vascular network that develops in tumours. Because tumour hypoxia has been associated with a more aggressive phenotype and lower cure rate, there is a recognized need for a method of measuring tumour hypoxia that is suitable for widespread clinical use. The aim of the current study was to compare the expression of Glut-1 with the binding of the bioreductive hypoxia marker pimonidazole and to elucidate the characteristics and pitfalls when they are used as hypoxic markers. Materials and methods. In the study, SA-1 solid subcutaneous tumours in A/J mice were treated by bleomycin given i.v. (1 mg/kg), or the application of electric pulses (8 pulses, 1400 V, 100 μs, 1Hz), or a combination of the two - electrochemotherapy. Pimonidazole was injected 16 hours before tumour excision. Tumours were excised at different time points (0.5, 1, 2, 8, 14 and 24 hours) after therapy. Immunohistochemistry for Glut-1 and pimonidazole adduct was carried out on two consecutive tumour sections and the percentages of positive staining areas were determined. Results. Glut-1 staining was membranous and typically expressed peri-necrotically, whereas pimonidazole staining, although showing substantial co-localisation with Glut-1, was cytoplasmatic. More than 65% of the stained areas showed a high degree of colocalization when the two markers were compared. Our results show that Glut-1 expression significantly correlates with the level of pimonidazole binding (r = 0.41; p = 0.028). Conclusions. Our study confirms that HIF-1 regulated genes, such as Glut-1, have potential for future use as predictors of a decreased sensitivity of tumours to radio- and chemotherapy mediated by hypoxia.

Cell culture chips for simultaneous application of topographical and electrical cues enhance phenotype of cardiomyocytes.

In vivo, cardiomyocytes are exposed to multiple biochemical and physical cues including topographical and electrical cues. During prolonged in vitro cultivation in standard tissue culture set-ups, cardiomyocytes are known to de-differentiate due to the lack of appropriate micro-environmental cues. Most currently available cell culture systems provide only a single biophysical cue, thus development of advanced cell cultivation systems incorporating multiple cues is urgently needed. We report here the development of a microfabricated system, incorporating topographical and electrical cues on a single chip, which enables cultivation of differentiated cardiomyocytes. The cell culture chips were created by hot embossing of polystyrene, to create microgrooves and microridges of precisely defined depth, width and periodicity. Substrates consisting of 0.5 microm-wide grooves and 0.5 microm-wide ridges (1 microm period) and those consisting of 3 microm-wide grooves and 1 microm-wide ridges (4 microm period) were investigated, with smooth surfaces used as controls. The depth of the microgrooves was 400 nm. The two gold electrodes were electrodeposited 1 cm apart such that the microgrooves in-between were oriented either parallel or perpendicular to the electrodes, enabling studies of interaction between topographical and electrical cues. Neonatal rat cardiomyocytes cultivated on microgrooved substrates for 7 days were elongated and aligned along the microgrooves forming a well developed contractile apparatus, as evidenced by sarcomeric alpha-actinin staining, with a more pronounced effect on substrates with 1 microm compared to 4 microm periodicity. Importantly, simultaneous application of biphasic electrical pulses and topographical cues resulted in gap junctions confined to the cell-cell end junctions rather than the punctate distribution found in neonatal cells. Electrical field stimulation further enhanced cardiomyocyte elongation when microgrooves were oriented parallel to the electric field. Due to the compatibility of the described cell culture chips with fluorescence and optical microscopy as well as the ability to independently control field stimulation parameters, biochemical and topographical cues on each chip, this system may in the future become a useful tool in drug development and maturation of cardiomyocytes derived from stem cells.

Enhanced cytotoxicity of bleomycin and cisplatin after electroporation in murine colorectal carcinoma cells

Background. Electrochemotherapy is a local treatment combining application of electric pulses and chemotherapy. Two chemotherapeutic drugs, bleomycin and cisplatin, have proved to be effective in electrochemotherapy. The effectiveness of electrochemotherapy was demonstrated in the treatment of various cutaneous and subcutaneous tumours in cancer patients. Only a few preclinical studies were performed in colorectal carcinoma, mostly using bleomycin. Our aim was to evaluate the sensitivity of the murine colorectal carcinoma cell line CMT-93 to electrochemotherapy with bleomycin and cisplatin for potential use in preclinical and clinical studies. Methods. CMT-93 cells were exposed to either the chemotherapeutic drug alone or electrochemotherapy. A clonogenic assay was used to determine cell survival after treatment. Apoptosis was measured by caspase- 3/7 activity, necrosis by changes in cell morphology and cell viability by the MTS assay 16 hours after electrochemotherapy. Results. Cells treated with electrochemotherapy were 500-fold more sensitive to bleomycin and 2.8-fold more sensitive to cisplatin compared to cells treated with the drugs alone. At the highest concentrations, a significant reduction in cell viability, increase in caspase-3/7 activity and necrotic cells were observed after electrochemotherapy. Conclusions. Exposure of cells to electric pulses enhanced cytotoxicity of both bleomycin and cisplatin. Reduced cell viability was due to apoptotic and necrotic cell death. Furthermore, electrochemotherapy with bleomycin was more cytotoxic than electrochemotherapy with cisplatin in this colorectal carcinoma cell line.

Non-volatile resistive switching in the dielectric superconductorYBa2Cu3O7−δ

We report on the reversible, non-volatile and polarity-dependent resistiveswitching between superconductor and insulator states at the interfaces of anAu/YBa2Cu3O7−δ (YBCO)/Au system. We show that, upon application of electric pulses, the superconductingstate of YBCO in regions near the electrodes can be reversibly removed and restored. Inaddition, four-wire measurements reveal that pulsing also induces significant non-volatilechanges in the bulk resistance. We argue that our observations are compatiblewith a scenario where the switching effect is due to migration of oxygen ionsalong grain boundaries that control the inter-grain superconducting coupling.

Electrochemotherapy of Tumours

Electrochemotherapy is a combined use of certain chemotherapeutic drugs and electric pulses applied to the treated tumour nodule. Local application of electric pulses to the tumour increases drug delivery into cells, specifically at the site of electric pulse application. Drug uptake by delivery of electric pulses is increased for only those chemotherapeutic drugs whose transport through the plasma membrane is impeded. Among many drugs that have been tested so far, bleomycin and cisplatin found their way from preclinical testing to clinical use. Clinical data collected within a number of clinical studies indicate that approximately 80% of the treated cutaneous and subcutaneous tumour nodules of different malignancies are in an objective response, from these, approximately 70% in complete response after a single application of electrochemotherapy. Usually only one treatment is needed, however, electrochemotherapy can be repeated several times every few weeks with equal effectiveness each time. The treatment results in an effective eradication of the treated nodules, with a good cosmetic effect without tissue scarring.

Electrochemotherapy of Tumours

Electrochemotherapy is a combined use of certain chemotherapeutic drugs and electric pulses applied to the treated tumour nodule. Local application of electric pulses to the tumour increases drug delivery into cells, specifically at the site of electric pulse application. Drug uptake by delivery of electric pulses is increased for only those chemotherapeutic drugs whose transport through the plasma membrane is impeded. Among many drugs that have been tested so far, bleomycin and cisplatin found their way from preclinical testing to clinical use. Clinical data collected within a number of clinical studies indicate that approximately 80% of the treated cutaneous and subcutaneous tumour nodules of different malignancies are in an objective response, from these, approximately 70% in complete response after a single application of electrochemotherapy. Usually only one treatment is needed, however, electrochemotherapy can be repeated several times every few weeks with equal effectiveness each time. The treatment results in an effective eradication of the treated nodules, with a good cosmetic effect without tissue scarring.

What is (Still not) Known of the Mechanism by Which Electroporation Mediates Gene Transfer and Expression in Cells and Tissues

Cell membranes can be transiently permeabilized under application of electric pulses. This treatment allows hydrophilic therapeutic molecules, such as anticancer drugs and DNA, to enter into cells and tissues. This process, called electropermeabilization or electroporation, has been rapidly developed over the last decade to deliver genes to tissues and organs, but there is a general agreement that very little is known about what is really occurring during membrane electropermeabilization. It is well accepted that the entry of small molecules, such as anticancer drugs, occurs mostly through simple diffusion after the pulse while the entry of macromolecules, such as DNA, occurs through a multistep mechanism involving the electrophoretically driven interaction of the DNA molecule with the destabilized membrane during the pulse and then its passage across the membrane. Therefore, successful DNA electrotransfer into cells depends not only on cell permeabilization but also on the way plasmid DNA interacts with the plasma membrane and, once into the cytoplasm, migrates towards the nucleus. The focus of this review is to describe the different aspects of what is known of the mechanism of membrane permeabilization and associated gene transfer and, by doing so, what are the actual limits of the DNA delivery into cells.

OcuDrain-E™—A noninvasive technique for reduction of intraocular pressure

OcuDrain-E™ is a noninvasive technique in which electrical pulses are applied across the cornea to enhance the rate of transcorneal water evaporation (TCWE). In vitro studies were carried out with rabbit cornea mounted on a Franz diffusion cell. Application of 30 pulses each of 1 millisecond (ms) duration at ≥40 V/cm 2 decreased the corneal resistivity ∼80% indicating permeabilization of the cornea. The corneal resistivity was almost completely recovered within 6 h when the pulse voltage was <40 V/cm 2. The average TCWE at 40 V/cm 2 was significantly (∼39-fold) higher than the control ( t-test, p < 0.0001). Application of electrical pulses (40 V–30 pulses–1 ms–1 Hz) across the cornea resulted in significant decrease in the intraocular pressure (IOP) in rabbits. The electrical protocol was well tolerated by the rabbits. Microscopic studies revealed that the applied electrical protocol did not cause any edema or detachment of the epidermal layers. The results of current investigation suggest that OcuDrain-E™ could be developed as a potential technique for the treatment of glaucoma in patients who respond poorly to drugs.

Numerical Models of Skin Electropermeabilization Taking Into Account Conductivity Changes and the Presence of Local Transport Regions

The protective function of skin (its low permeability) presents a formidable obstacle in therapeutical applications such as transdermal drug delivery and gene delivery in skin. One of the possibilities to temporarily breach the barrier function of skin is using electroporation, creating aqueous pathways across lipid-based structures by means of electric pulses. In addition, the application of electric pulses to biological cells causes the electroporation of cell membrane, increasing its permeability, thus enabling cell uptake of larger molecules that otherwise cannot cross the membrane, such as drug molecules or DNA. The electropermeabilization process in skin was described theoretically, by means of numerical modeling, leaning on data derived from our in vivo experiments previously published. The numerical models took into account the layered structure of skin, macroscopical changes of its bulk electrical properties during electroporation, as well as the presence of localized sites of increased molecular transport termed local transport regions. The output of the models was compared with the in vivo experiments, and a good agreement was obtained. In addition, a comparison of our results with already published findings on skin electropermeabilization showed that permeabilizing voltage amplitudes suggested by the model are also well in the range of the voltage amplitudes reported by other authors to cause skin permeabilization. The subject of tissue conductivity changes due to electroporation is still a rather unexplored field; however, we used the available data to describe the mechanism of the nonlinear process of the tissue electropermeabilization.

Electrochemotherapy in Veterinary Oncology

Electropermeabilization is a method that uses electric field pulses to induce an electrically mediated reorganization of the plasma membrane of cells. Electrochemotherapy combines local or systemic administration of chemotherapeutic drugs such as bleomycin or cisplatin that have poor membrane permeability with electropermeabilization by direct application of electric pulses to the tumors. Preclinical studies have demonstrated excellent antitumor effectiveness of electrochemotherapy on different animal models and various tumor types, minimal toxicity, and safety of the procedure. Based on results of preclinical studies, clinical studies were conducted in human patients, which demonstrated pronounced antitumor effectiveness of electrochemotherapy with 80-85% objective responses of the treated cutaneous and SC tumors. Clinical studies in veterinary oncology have demonstrated that electrochemotherapy is very effective in the treatment of cutaneous and SC tumors of different histologic types in cats, dogs, and horses. The results of these studies have also demonstrated approximately 80% long-lasting objective responses of tumors treated by electrochemotherapy. Primary tumors of different histologic types were treated. Electrochemotherapy in veterinary oncology has future promise to be highly effective, and could be used to treat primary or recurrent solitary or multiple cutaneous and SC tumors of different histology or as an adjuvant treatment to surgery.

A Numerical Model of Permeabilized Skin With Local Transport Regions

The protective function of skin and hence its low permeability presents a formidable obstacle in therapeutical applications such as transdermal drug delivery and gene delivery in skin. One of the methods to temporarily increase skin permeability is electroporation, creating aqueous pathways across lipid-based structures by means of electric pulses. Also, the application of electric pulses to biological cells causes increased permeability of cell membrane, thus enabling the uptake of larger molecules that otherwise cannot cross the membrane, such as drug molecules or DNA, into the cell. The creation of localized sites of increased molecular transport termed local transport regions (LTRs) can be observed during electroporation, as well as changes in the bulk electric properties of skin layers. We modeled these phenomena with a numerical model and compared the output of the model with our own in vivo experiments and previously published results of skin electroporation and a good agreement was obtained. With the model presented, we used the available data to describe the nonlinear process of skin electropermeabilization from the aspect of tissue conductivity changes and the presence of local transport regions in the permeabilized stratum corneum. The observations derived from various in vivo experiments by different authors were thus confirmed theoretically.

Studies on mRNA Electroporation of Immature and Mature Dendritic Cells: Effects on their Immunogenic Potential

Previous studies have shown that mRNA-electroporated dendritic cells (DCs) are able to process and present tumor-associated antigens, leading to the activation of tumor-specific T cells in vitro and in vivo. However, the optimal maturation state of antigen loading and half-life of the mRNA-translated protein product and its immunogenic epitopes are significant parameters, which needs to be clarified in order to establish an effective electroporation protocol. In addition, despite extensive experimental investigations and their widespread application in research and clinical environments, little is known of the extent to which the immunological properties of DCs are influenced by electrical fields of critical strengths. We found that the mRNA transfection of DCs after maturation with short and low-voltage square-wave electrical pulses resulted in higher level of antigen expression and viability in addition to higher T-cell stimulatory ability compared to transfection of DCs prior to maturation. Mature mRNA-electroporated DCs showed long-lived expression of EGFP and were able to stimulate influenza matrix protein M1 (M1)-specific T cells up to 24 h after electroporation. However, when DCs were subjected to increasing electrical pulses the level of transgene expression was four-fold upregulated, equipping these DCs to be more potent in inducing M1-specific T cells. Also, the application of long electrical pulses induced further upregulation of HLA-DR, CD80, and CD86 expression in mature DCs, but did not promote phenotypic or functional maturation in immature DCs. These findings support the concept of mRNA transfection of DCs after maturation and also highlight the possibility to use long electrical pulses for further improvement of the immune responses by mRNA-transfected DCs.

ZAP-70 Restoration in Mice by In Vivo Thymic Electroporation

Viral and non-viral vectors have been developed for gene therapy, but their use is associated with unresolved problems of efficacy and safety. Efficient and safe methods of DNA delivery need to be found for medical application. Here we report a new monopolar system of non-viral electro-gene transfer into the thymus in vivo that consists of the local application of electrical pulses after the introduction of the DNA. We assessed the proof of concept of this approach by correcting ZAP-70 deficient severe combined immunodeficiency (SCID) in mice. The thymic electro-gene transfer of the pCMV-ZAP-70-IRES-EGFP vector in these mice resulted in rapid T cell differentiation in the thymus with mature lymphocytes detected by three weeks in secondary lymphoid organs. Moreover, this system resulted in the generation of long-term functional T lymphocytes. Peripheral reconstituted T cells displayed a diversified T cell receptor (TCR) repertoire, and were responsive to alloantigens in vivo. This process applied to the thymus could represent a simplified and effective alternative for gene therapy of T cell immunodeficiencies.

The temperature effect during pulse application on cell membrane fluidity and permeabilization

Cell membrane permeabilization is caused by the application of high intensity electric pulses of short duration. The extent of cell membrane permeabilization depends on electric pulse parameters, characteristics of the electropermeabilization media and properties of cells exposed to electric pulses. In the present study, the temperature effect during pulse application on cell membrane fluidity and permeabilization was determined in two different cell lines: V-79 and B16F-1.While cell membrane fluidity was determined by electron paramagnetic resonance (EPR) method, the cell membrane electropermeabilization was determined by uptake of bleomycin and clonogenic assay. A train of eight rectangular pulses with the amplitude of 500 V/cm, 700 V/cm and 900 V/cm in the duration of 100 μs and with repetition frequency 1 Hz was applied. Immediately after the pulse application, 50 μl droplet of cell suspension was maintained at room temperature in order to allow cell membrane resealing. The cells were then plated for clonogenic assay. The main finding of this study is that the chilling of cell suspension from physiological temperature (of 37 °C) to 4 °C has significant effect on cell membrane electropermeabilization, leading to lower percent of cell membrane permeabilization. The differences are most pronounced when cells are exposed to electric pulse amplitude of 900 V/cm. At the same time with the decreasing of temperature, the cell membranes become less fluid, with higher order parameters in all three types of domains and higher proportion of domain with highest order parameter. Our results indicate that cell membrane fluidity and domain structure influence the electropermeabilization of cells, however it seems that some other factors may have contributing role.

Gene Electrotransfer into Murine Skeletal Muscle: A Systematic Analysis of Parameters for Long-term Gene Expression

Skeletal muscle is an attractive target tissue for delivery of therapeutic genes, since it is well vascularized, easily accessible, and has a high capacity for protein synthesis. For efficient transfection in skeletal muscle, several protocols have been described, including delivery of low voltage electric pulses and a combination of high and low voltage electric pulses. The aim of this study was to determine the influence of different parameters of electrotransfection on short-term and long-term transfection efficiency in murine skeletal muscle, and to evaluate histological changes in the treated tissue. Different parameters of electric pulses, different time lags between plasmid DNA injection and application of electric pulses, and different doses of plasmid DNA were tested for electrotransfection of tibialis cranialis muscle of C57Bl/6 mice using DNA plasmid encoding green fluorescent protein (GFP). Transfection efficiency was assessed on frozen tissue sections one week after electrotransfection using a fluorescence microscope and also noninvasively, followed by an in vivo imaging system using a fluorescence stereo microscope over a period of several months. Histological changes in muscle were evaluated immediately or several months after electrotransfection by determining infiltration of inflammatory mononuclear cells and presence of necrotic muscle fibers. The most efficient electrotransfection into skeletal muscle of C57Bl/6 mice in our experiments was achieved when one high voltage (HV) and four low voltage (LV) electric pulses were applied 5 seconds after the injection of 30 microg of plasmid DNA. This protocol resulted in the highest short-term as well as long-term transfection. The fluorescence intensity of the transfected area declined after 2-3 weeks, but GFP fluorescence was still detectable 18 months after electrotransfection. Extensive inflammatory mononuclear cell infiltration was observed immediately after the electrotransfection procedure using the described parameters, but no necrosis or late tissue damage was observed. This study showed that electric pulse parameters, time lag between the injection of DNA and application of electric pulses, and dose of plasmid DNA affected the duration of transgene expression in murine skeletal muscle. Therefore, transgene expression in muscle can be controlled by appropriate selection of electrotransfection protocol.

RELATIVE ELECTRO PERMEABILITY AND ELECTRIC PULSE EFFECTIVENESS IN HUMAN BREAST ADEN CARCINOMA: AN IN VITRO STUDY

Objective(s) This study was carried out in order to evaluate the effects of electrochemotherapy, electrical pulses and chemotherapeutic drugs on the killing of cancerous cells and their probable synergistic effects. Materials and Methods Electrochemotherapy treatments conducted on MCF-7 cell line derived from human breast adenocarcinoma tumor using four chemotherapeutic drugs including bleomycin, cisplatin, adriamycin and cyclophosphamide and six electrical doses. Cell survival assayed using MTT method, 72 hrs after the treatment; also the killing effects of each drug and electric dose determined. Finally, “Relative Pulse Effectiveness” and “Relative Electropermeability Effectiveness” calculated. Results All electrical doses decreased cell survival, significantly for bleomycin and cisplatin, however, they were only, significant in high concentration of cyclophosphamide and adriamycin. For the applied drugs, “Relative Electropermeability Effectiveness” was more than one (1.00), except for adriamycin. Conclusion It seems that for the diffusion of molecules into cells, application of high duration electric pulses is more efficient for high molecular weight drugs while for low molecular weight drugs, strong pulses are more effective. In intermediate molecular weight, there is no difference between increasing the pulse strength and/or duration to achieve additional electropermeability. Electropermeability effect of different electric doses and electrochemotherapy efficiency can be evaluated by “REE” and “RPE”, respectively.