2-ns Electrostimulation of Ca2+ Influx into Chromaffin Cells: Rapid Modulation by Field Reversal

Cellular effects of nanosecond-pulsed electric field exposures can be attenuated by an electric field reversal, a phenomenon called bipolar pulse cancellation. Our investigations of this phenomenon in neuroendocrine adrenal chromaffin cells show that a single 2-ns, 16 MV/m unipolar pulse elicited a rapid, transient rise in intracellular Ca²⁺ levels due to Ca²⁺ influx through voltage-gated calcium channels. The response was eliminated by a 2-ns bipolar pulse with positive and negative phases of equal duration and amplitude and fully restored (unipolar-equivalent response) when the delay between each phase of the bipolar pulse was 30 ns. Longer interphase intervals evoked Ca²⁺ responses that were greater in magnitude than those evoked by a unipolar pulse (stimulation). Cancellation was also observed when the amplitude of the second (negative) phase of the bipolar pulse was half that of the first (positive) phase but progressively lost as the amplitude of the second phase was incrementally increased above that of the first phase. When the amplitude of the second phase was twice that of the first phase, there was stimulation. By comparing the experimental results for each manipulation of the bipolar pulse waveform with analytical calculations of capacitive membrane charging/discharging, also known as accelerated membrane discharge mechanism, we show that the transition from cancellation to unipolar-equivalent stimulation broadly agrees with this model. Taken as a whole, our results demonstrate that electrostimulation of adrenal chromaffin cells with ultrashort pulses can be modulated with interphase intervals of tens of nanoseconds, a prediction of the accelerated membrane discharge mechanism not previously observed in other bipolar pulse cancellation studies. Such modulation of Ca²⁺ responses in a neural-type cell is promising for the potential use of nanosecond bipolar pulse technologies for remote electrostimulation applications for neuromodulation.     

Inactivation of Pseudomonas putida by Pulsed Electric Field Treatment: A Study on the Correlation of Treatment Parameters and Inactivation Efficiency in the Short-Pulse Range

An important issue for an economic application of the pulsed electric field treatment for bacterial decontamination of wastewater is the specific treatment energy needed for effective reduction of bacterial populations. The present experimental study performed in a field amplitude range of 40 > E > 200 kV/cm and for a suspension conductivity of 0.01 = κ _e > 0.2 S/m focusses on the application of short pulses, 25 ns > T > 10 μs, of rectangular, bipolar and exponential shape and was made on Pseudomonas putida, which is a typical and widespread wastewater microorganism. The comparison of inactivation results with calculations of the temporal and azimuthal membrane charging dynamics using the model of Pauly and Schwan revealed that for efficient inactivation, membrane segments at the cell equator have to be charged quickly and to a sufficiently high value, on the order of 0.5 V. After fulfilling this basic condition by an appropriate choice of pulse field strength and duration, the log rate of inactivation for a given suspension conductivity of 0.2 S/m was found to be independent of the duration of individual pulses for constant treatment energy expenditure. Moreover, experimental results suggest that even pulse shape plays a minor role in inactivation efficiency. The variation of the suspension conductivity resulted in comparable inactivation performance of identical pulse parameters if the product of pulse duration and number of pulses was the same, i.e., required treatment energy can be linearly downscaled for lower conductivities, provided that pulse amplitude and duration are selected for entire membrane surface permeabilization.     

Modulation of biological responses to 2 ns electrical stimuli by field reversal

Nanosecond bipolar pulse cancellation, a recently discovered phenomenon, is modulation of the effects of a unipolar electric pulse exposure by a second pulse of opposite polarity. This attenuation of biological response by reversal of the electric field direction has been reported with pulse durations from 60 ns to 900 ns for a wide range of endpoints, and it is not observed with conventional electroporation pulses of much longer duration (>100 μs) where pulses are additive regardless of polarity. The most plausible proposed mechanisms involve the field-driven migration of ions to and from the membrane interface (accelerated membrane discharge). Here we report 2 ns bipolar pulse cancellation, extending the scale of previously published results down to the time required to construct the permeabilizing lipid electropores observed in molecular simulations. We add new cancellation endpoints, and we describe new bipolar pulse effects that are distinct from cancellation. This new data, which includes transport of cationic and anionic permeability indicators, fluorescence of membrane labels, and patterns of entry into permeabilized cells, is not readily explained by the accelerated discharge mechanism. We suggest that multi-step processes that involve first charged species movement and then responses of cellular homeostasis and repair mechanisms are more likely to explain the broad range of reported results.     

Permeabilization of cell membrane by programmed form of electrical pulses

The role of the shape of electric pulses of cell permeabilization and lysis was studied using the newly developed DPS electroporator. The effects of bipolar pulses, steep rising and falling edges in the pulses, delays between pulses, and shapes of DC signals between the edges on the lysis of bovine oocytes and the permeabilization of their cell membranes were investigated. Comparing the permeabilization rates with the lysis rates revealed a number of correlations, which make it possible to optimize the pulse shapes for achieving maximum permeabilization rates while keeping the lysis rates low. The optimization of pulse shape is essential for improving the procedure of electroporation in mammalian cloning technology.     

Optimization of Electric Pulse Amplitude and Frequency In Vitro for Low Voltage and High Frequency Electrochemotherapy

During standard electrochemotherapy (ECT), using a train of 1,000 V/cm amplitude rectangular pulses with 1 Hz frequency, patients experience an unpleasant sensation and slight edema. According to the patients, muscle contractions provoked by high amplitude (about 1,000 V/cm) and low repetition frequency (1 Hz) pulses are the most unpleasant and painful sensations. Recently, ECT using low voltage and higher repetition frequency (LVHF) has been shown to be an effective tool for inhibiting tumor growth. The aim of the present study was to optimize electric pulse amplitude and repetition frequency for LVHF ECT by sampling the different sets of pulse parameters on cell viability and permeabilization. In ECT, a reversible effect based on high permeabilization is desirable. For this purpose, we used bleomycin to evaluate the permeabilization of K562 and MIA-PACA2 cells caused by low voltage (50–150 V/cm) and higher repetition frequency (4–6 kHz) electric pulses. We show that the reversible effect with electropermeabilization of the cells caused by LVHF ECT is accessible; this interaction is more effective for electric pulses with 70 V/cm amplitude.     

Effect of stimulus (postsynaptic current) shape on fibre excitation.

Effects of variation of the stimulus pulse shape on the excitation of a nonmyelinated nerve fibre were studied using a mathematical model based on the Hodgkin-Huxley equations. Efficiency of smoothly changing pulses was compared with that of rectangular pulses. For pulses shorter than the time to excitation, the rate of the stimulus rise did not determine the ability of a smoothly changing pulse to excite the fibre. For a given stimulus duration, the main factor was the pulse area or the charge delivered by the pulse. The strength-duration curve for smoothly changing pulses was a nonmonotonic function, in contrast to the curve for rectangular pulses. The dependence of latency on changes in the pulse area was non-linear. It would be nonmonotonic when the pulse area variation were due to the stimulus duration or the stimulus rise duration. More that one propagating intracellular action potential (IAP) could arise upon fibre activation by a long smoothly changing threshold stimulus. Upon activation of relatively short fibres the IAP could arise not at the site of the smoothly changing stimulus injection. The rectangular pulses of long duration were more efficient than the corresponding smoothly changing ones. Irrespective of the shape, the pulses whose duration at the foot is 1-2 ms, are more suitable for a prolonged threshold fibre activation.     

一种改进的杂交瘤细胞电融合方法

一种改进的杂交瘤细胞电融合方法邹翔（南京大学医学院南京２１０００８）刘琴芝,裴红英,郁文芳,安节,陈伯权（中国预防医学科学院病毒学研究所北京１０００５２）陈刚,赵南明（清华大学生物科学与技术系生物膜与膜生物工程国家重点实验室北京１０００８４）我们对国...     

Electric Field Orientation for Gene Delivery Using High-Voltage and Low-Voltage Pulses

Electropermeabilization is a biological physical process in response to the presence of an applied electric field that is used for the transfer of hydrophilic molecules such as anticancer drugs or DNA across the plasma membranes of living cells. The molecular processes that support the transfer are poorly known. The aim of our study was to investigate the effect of high-voltage and low-voltage (HVLV) pulses in vitro with different orientations on cell permeabilization, viability and gene transfection. We monitored the permeabilization with unipolar and bipolar HVLV pulses with different train repetition pulses, showing that HVLV pulses increase cell permeabilization and cell viability. Gene transfer was also observed by measuring green fluorescent protein (GFP) expression. The expression was the same for HVLV pulses and electrogenotherapy pulses for in vitro experimentation. As the viability was better preserved for HVLV-pulsed cells, we managed to increase the number of GFP-expressing cells by up to 65?% under this condition. The use of bipolar HVLV train pulses increased gene expression to a higher extent, probably by affecting a larger part of the cell surface.     

Membrane voltage changes in a compartmental chain model of a neurone

A mathematical model of the neurone has been developed using the method of subdivision of the neurone into a number of equivalent circuit compartments. Compartmental characteristics have been investigated by calculating the shape indices of the output produced in response to a given somatic input conductance change. A generalised form of compartmental chain has been chosen to allow calculation of the shape indices produced by a variety of geometrical configurations including the straight and tapering chain forms. Equations have been deduced from the computations made on a CDC 6600 computer relating the peak amplitude of the output response to the compartmental diameter for both the straight and tapering chain forms. The effect of variation in the location of the input conductance injection site has also been related to the peak amplitude of the somatic response. The optimum characteristics of the input conductance pulse shape have been computed initially using a rectangular pulse and later the more physiologically relevant double exponential shape. The effect of alteration in the end compartmental terminal impedances over the range from open to short circuit conditions was also calculated. The establishment of optimum single compartmental chain criteria allows the future investigation of multiple chain and pyramidal cell configurations.     

Solid state NMR at high magic angle spinning frequencies: Dipolar chemical shift correlation with adiabatic inversion pulse based RF pulse schemes

The efficacy of hetero- and homonuclear dipolar recoupling employing tanh/tan adiabatic inversion pulse based RF pulse schemes has been examined at high magic angle spinning (MAS) frequencies via numerical simulations and experimental measurements. An approach for minimising the recoupling RF power level is presented, taking into consideration the spinning speed, the range of resonance offsets and H₁ inhomogeneities and the available RF field strength. This involves the tailoring of the frequency and amplitude modulation profiles of the inversion pulses. The applicability of tanh/tan pulse based dipolar recoupling schemes to spinning speed regimes where the performance with conventional rectangular pulses may not be satisfactory is demonstrated.     

Analysis and Optimization of Pulse Dynamics for Magnetic Stimulation

Magnetic stimulation is a standard tool in brain research and has found important clinical applications in neurology, psychiatry, and rehabilitation. Whereas coil designs and the spatial field properties have been intensively studied in the literature, the temporal dynamics of the field has received less attention. Typically, the magnetic field waveform is determined by available device circuit topologies rather than by consideration of what is optimal for neural stimulation. This paper analyzes and optimizes the waveform dynamics using a nonlinear model of a mammalian axon. The optimization objective was to minimize the pulse energy loss. The energy loss drives power consumption and heating, which are the dominating limitations of magnetic stimulation. The optimization approach is based on a hybrid global-local method. Different coordinate systems for describing the continuous waveforms in a limited parameter space are defined for numerical stability. The optimization results suggest that there are waveforms with substantially higher efficiency than that of traditional pulse shapes. One class of optimal pulses is analyzed further. Although the coil voltage profile of these waveforms is almost rectangular, the corresponding current shape presents distinctive characteristics, such as a slow low-amplitude first phase which precedes the main pulse and reduces the losses. Representatives of this class of waveforms corresponding to different maximum voltages are linked by a nonlinear transformation. The main phase, however, scales with time only. As with conventional magnetic stimulation pulses, briefer pulses result in lower energy loss but require higher coil voltage than longer pulses.     

Optimization of electroporation for transfection of mammalian cell lines

Electroporation can be a highly efficient method for introducing DNA molecules into cultured cells for transient expression of genes or for permanent genetic modification. However, effective transformation by electroporation requires careful optimization of electric field strength and pulse characteristics. We have used the transient expression of the firefly luciferase gene as a rapid and sensitive indicator of gene expression to describe the effects on transfection efficiency of altering electroporation field strength and shape. Using the luciferase assay, we investigated the correlation of cell viability with optimal transfection efficiency and determined the optimal parameters for a number of phenotypically distinct mammalian cell lines derived from the nervous and immune systems. The efficiency of electroporation under optimal conditions was compared with that obtained using DEAE-dextran or calcium phosphate-mediated transformation. Transfection by electroporation using square wave pulses, as opposed to exponentially decaying pulses, was found to be significantly increased by repetitive pulses. These methods improve the ability to obtain high efficiency gene transfer into many mammalian cell types.     

Unipolar and bipolar electrogram characteristics of recurrent cases of idiopathic ventricular arrhythmias undergoing repeat catheter ablation

IntroductionActivation mapping guided catheter ablation (CA) of ventricular arrhythmias (VAs) is limited in some cases when it is only relied on bipolar electrogram (EGM). We hypothesized that activation mapping with use of combined bipolar and unipolar EGM facilitates to identify the focal origin of VAs and results in reduction of recurrence rate of CA of VAs.MethodsWe analyzed the data of patients undergoing repeat ablations for idiopathic out-flow tract VAs. The EGM of the 1 st and 2 nd ablations were compared for earliest local activation time (LAT), presence of discrete potentials, and polarity reversal, unipolar potential morphology (QS or non-QS), potential amplitude and activation slope.ResultsThirty-seven patients were included. The Local activation time was significantly earlier in the 2nd ablation as compared to the 1st procedure (36.90 msec vs 31.85 msec, P < 0.01). The incidence of discrete potentials and polarity reversal were similar in both procedures (51% vs 57%, P = 0.8 and 62% in both the occasions, respectively). The unipolar voltage was similar in both occasions (6.94 mV vs 7.22 mV in repeat ablations, P = 0.7). The recurrence rate (5.7%) was significantly lower with routine use of combined unipolar and bipolar EGMs, as compared to the use of bipolar EGM alone (16.7%)ConclusionsUse of both bipolar and unipolar electrograms helps in better delineation of the sites of earliest activation for effective ablation of VAs. Use of unipolar electrograms in addition to bipolar electrograms is associated with lower long term recurrence rate.     

Direct gene transfer to plant protoplasts by electroporation by alternating,rectangular and exponentially decaying pulses

Summary The efficiency of electroporation of sugar beet (Beta vulgaris L.) and tobacco (Nicotiana tabacum L.) protoplasts by alternating, rectangular and exponentially decaying pulses was studied by assaying transient expression of an introduced gene for chloramphenicol acetyltransferase. A simple device for electroporation by alternating current was constructed. The mains (220 V) were used as power supply and the pulse duration was controlled by the blow-out of a small fuse. Electroporation of sugar beet protoplasts by alternating current and exponentially decaying pulses resulted in 3–4 fold higher transient expression compared to rectangular pulses. Transient expression in tobacco protoplasts electroporated by exponentially decaying pulses was 30 % and 85 % higher than when electroporated by rectangular and alternating current pulses, respectively.Abbreviations CAT Chloramphenicol acetyltransferase     

Increased Neural Cell Adhesion Molecule in the CSF of Patients with Mood Disorder

Abstract: Neural cell adhesion molecule (N-CAM) is involved in cell-cell interactions during synaptogenesis, morphogenesis, and plasticity of the nervous system. Disturbances in synaptic restructuring and neural plasticity may be related to the pathogenesis of several neuropsychiatric diseases, including mood disorders and schizophrenia. Disturbances in brain cellular function may alter concentrations of N-CAM in the CSF. Soluble human N-CAM proteins are detectable in the CSF but are minor constituents of serum. We have recently found an increase in N-CAM content in the CSF of patients with schizophrenia. Although the pathogenesis of both schizophrenia and mood disorders is unknown, ventriculomegaly, decreased temporal lobe volume, and subcortical structural abnormalities have been reported for both disorders. We have therefore measured N-CAM concentrations in the CSF of patients with mood disorder. There were significant increases in amounts of N-CAM immunoreactive proteins, primarily the 120-kDa band, in the CSF of psychiatric inpatients with bipolar mood disorder type I and recurrent unipolar major depression. There were no differences in bipolar mood disorder type II patients as compared with normals. There were no significant effects of medication treatment on N-CAM concentrations. It is possible that the 120-kDa N-CAM band present in the CSF is derived from CNS cells as a secreted soluble N-CAM isoform. Our results suggest the possibility of latent state-related disturbances in N-CAM cellular function, i.e., residue from a previous episode, or abnormal N-CAM turnover in the CNS of patients with mood disorder.     

On the molecular mechanisms implicated in the bipolar cancellation of membrane electroporation

Bipolar cancellation is the phenomenon in which the permeability of cell membranes subjected to high intensity short pulsed electric field (ns-μs range) is reduced or eliminated when the system is subjected to bipolar instead of monopolar pulses. Although several studies have tried to explain bipolar cancellation, the underlying mechanisms remain unclear. Very few articles study bipolar cancellation by means of molecular dynamics (MD) simulation. In this paper, we investigated the molecular mechanisms underlying the difference in electroporation induced by bipolar and monopolar picosecond electric pulses (EPs) using MD simulation. The electric field gradients and electric forces on water molecules of the two pulses were analyzed in detail for the first time. For a certain pulse width, when the field intensity is relatively small, the direction of bipolar electric force on the interfacial water molecule reverses as the bipolar EPs reverse, while the electric force on interfacial water molecules of the cathode side remains in the same direction as that of applied monopolar EPs. The bipolar electric force reversal delays the water protrusion and increases the pore formation time. Therefore, this phenomenon could correspond to bipolar cancellation. When the field intensity is relatively large, although the bipolar electric force direction still reverses, half of the total time of the monopolar EPs has no electric fields. The electric forces of monopolar no-field half-cycles are much smaller than those of the bipolar EPs. Therefore, the pore formation time of bipolar EPs reduces, and this phenomenon is called bipolar enhancement. The occurrence of bipolar cancellation or bipolar enhancement depends on conditions such as the width and intensity of the pulse.     

Modified Blumlein Pulse-Forming Networks for Bioelectrical Applications

Intense nanosecond pulsed electric fields (nsPEFs) have been shown to induce, on intracellular structures, interesting effects dependent on electrical exposure conditions (pulse length and amplitude, repetition frequency and number of pulses), which are known in the literature as “bioelectrical effects” (Schoenbach et al., IEEE Trans Plasma Sci 30:293–300, 2002). In particular, pulses with a shorter width than the plasma membrane charging time constant (about 100 ns for mammalian cells) can penetrate the cell and trigger effects such as permeabilization of intracellular membranes, release of Ca²⁺ and apoptosis induction. Moreover, the observed effects have led to exploration of medical applications, like the treatment of melanoma tumors (Nuccitelli et al., Biochem Biophys Res Commun 343:351–360, 2006). Pulsed electric fields allowing such effects usually range from several tens to a few hundred nanoseconds in duration and from a few to several tens of megavolts per meter in amplitude (Schoenbach et al., IEEE Trans Diel Elec Insul 14:1088–1109, 2007); however, the biological effects of subnanosecond pulses have been also investigated (Schoenbach et al., IEEE Trans Plasma Sci 36:414–422, 2008). The use of such a large variety of pulse parameters suggests that highly flexible pulse-generating systems, able to deliver wide ranges of pulse durations and amplitudes, are strongly required in order to explore effects and applications related to different exposure conditions. The Blumlein pulse-forming network is an often-employed circuit topology for the generation of high-voltage electric pulses with fixed pulse duration. An innovative modification to the Blumlein circuit has been recently devised which allows generation of pulses with variable amplitude, duration and polarity. Two different modified Blumlein pulse-generating systems are presented in this article, the first based on a coaxial cable configuration, matching microscopic slides as a pulse-delivery system, and the other based on microstrip transmission lines and designed to match cuvettes for the exposure of cell suspensions.     

CHROMOSOME MICROMANIPULATION : III. Spindle Fiber Tension and the Reorientation of Mal-Oriented Chromosomes

Kinetochore reorientation is the critical process ensuring normal chromosome distribution. Reorientation has been studied in living grasshopper spermatocytes, in which bivalents with both chromosomes oriented to the same pole (unipolar orientation) occur but are unstable: sooner or later one chromosome reorients, the stable, bipolar orientation results, and normal anaphase segregation to opposite poles follows. One possible source of stability in bipolar orientations is the normal spindle forces toward opposite poles, which slightly stretch the bivalent. This tension is lacking in unipolar orientations because all the chromosomal spindle fibers and spindle forces are directed toward one pole. The possible role of tension has been tested directly by micromanipulation of bivalents in unipolar orientation to artificially create the missing tension. Without exception, such bivalents never reorient before the tension is released; a total time "under tension" of over 5 hr has been accumulated in experiments on eight bivalents in eight cells. In control experiments these same bivalents reoriented from a unipolar orientation within 16 min, on the average, in the absence of tension. Controlled reorientation and chromosome segregation can be explained from the results of these and related experiments.     

Importance of association between permeabilization and electrophoretic forces for intramuscular DNA electrotransfer

Gene transfer using electrical pulses is a rapidly expanding field. Many studies have been performed in vitro to elucidate the mechanism of DNA electrotransfer. In vivo, the use of efficient procedures for DNA electrotransfer in tissues is recent, and the question of the implied mechanisms is largely open. We have evaluated the effects of various combinations of square wave electric pulses of variable field strength and duration, on cell permeabilization and on DNA transfection in the skeletal muscle in vivo. One high voltage pulse of 800 V/cm, 0.1 ms duration (short high pulse) or a series of four low voltage pulses of 80 V/cm, 83 ms duration (long low pulses) slightly amplified transfection efficacy, while no significant permeabilization was detected using the (51)Cr-EDTA uptake test. By contrast, the combination of one short high pulse followed by four long low pulses led to optimal gene transfer efficiency, while inducing muscle fibers permeabilization. These results are consistent with additive effects of electropermeabilization and DNA electrophoresis on electrotransfer efficiency. Finally, the described new combination, as compared to the previously reported use of repeated identical pulses of intermediate voltage, leads to similar gene transfer efficiency, while causing less permeabilization and thus being likely less deleterious. Thus, combination of pulses of various strengths and durations is a new procedure for skeletal muscle gene transfer that may represents a clear improvement in view of further clinical development.