2-ns Electrostimulation of Ca\u3csup\u3e2+\u3c/sup\u3e 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

Validation of Nanosecond Pulse Cancellation Using a Quadrupole Exposure System

Nanosecond pulsed electric fields (nsPEFs) offer a plethora of opportunities for developing integrative technologies as complements or alternatives to traditional medicine. Studies on the biological effects of nsPEFs in vitro and in vivo have revealed unique characteristics that suggest the potential for minimized risk of complications in patients, such as the ability of unipolar nsEPs to create permanent or transient pores in cell membranes that trigger localized lethal or non-lethal outcomes without consequential heating. A more recent finding was that such responses could be diminished by applying a bipolar pulse instead, a phenomenon dubbed bipolar cancellation, paving the way for greater flexibility in nsPEF application design. Transitioning nsPEFs into practical use, however, has been hampered by both device design optimization and the intricacies of mammalian biology. Generating electric fields capable of beneficially manipulating human physiology requires high-voltage electrical pulses of nanosecond duration (nsEPs) with high repetition rates, but pulse generator and electrode design in addition to the complex electrical properties of biological fluids and tissues dictate the strength range and distribution of the resulting electric field. Faced with both promising and challenging aspects to producing a biomedically viable option for inducing a desired nsPEF response that is both focused and minimally invasive, the question becomes: how can the distinct features of unipolar and bipolar nsPEF bioeffects be exploited in a complex electrode exposure system to spatially modulate cell permeabilization? This dissertation presents a systematic study of an efficient coplanar quadrupole electrode nsPEF delivery system that exploits unique differences between unipolar and bipolar nsPEF effects to validate its ability to control cell responses to nsPEFs in space. Four specific aims were established to answer the research question, with specific attention to the roles played by pulse polarity, grounding configuration and electric field magnitude in influencing nsPEF stimulation of electropermeabilization in space. Using a prototype wire electrode applicator charged by a custom-built multimodal pulse generator, the aims were to spatially quantifyelectropermeabilization due (1) unipolar and (2) bipolar nsPEF exposure, to (3) apply synchronized pulses with a view to canceling bipolar cancellation (CANCAN) through superposition that could shift the effective nsPEF response, and to (4) evaluate the ability of the quadrupole system to facilitate remote nsPEF electropermeabilization. Numerical simulations were employed to approximate the nsPEF distribution for a two-dimensional (2-D) area resulting from unipolar, bipolar or CANCAN exposure in a varied-pulse quadrupole electrode configuration. For all experiments, the independent variables were fixed for pulse width (600 ns), pulse number (50) and repetition rate (10 Hz). Electropermeabilization served as the biological endpoint, with green fluorescence due to cell uptake of the nuclear dye YO-PRO-1® (YP1) tracer molecule serving the response variable. An agarose-based 3-D tissue model was used to acquire, quantify and compare fluorescence intensity data in vitro, which was measured by stereomicroscopy to enable macro versus micro level 2-D visualization. Results of this investigation showed that increasing the magnitude of the applied voltage shifts unipolar responses from localization at the anodal to cathodal electrode, and that adding a second proximal ground electrode increases the response area. Bipolar nsPEF responses were generally less intense than unipolar, but these depended on both the inter-electrode location measured and amplitude of the second phase. CANCAN preliminary indicated some ability to decrease strong uptake at electrodes, but evaluation across experimental and published data indicate that greater differences between unipolar and bipolar responses are needed to improve possibilities for distal stimulation. Overall, this work demonstrated the potential for more complex pulser-electrode configurations to successfully modulate nsPEF electropermeabilization in space by controlling unipolar and bipolar pulse delivery and contributed to a deeper understanding of bipolar cancellation. By providing a set of metrics for test and evaluation, the data provided herein may serve to inform model development to support prediction of nsPEF outcomes and help to more acutely define spatial-intensity relationships between nsPEFs and cell permeabilization as well as delineate requirements for future non-invasive nsPEF therapies

Aerospace Medicine and Biology: A continuing bibliography (supplement 160)

This bibliography lists 166 reports, articles, and other documents introduced into the NASA scientific and technical information system in October 1976

Modeling Morphogenesis in silico and in vitro: Towards Quantitative, Predictive, Cell-based Modeling

Cell-based, mathematical models help make sense of morphogenesis—i.e. cells organizing into shape and pattern—by capturing cell behavior in simple, purely descriptive models. Cell-based models then predict the tissue-level patterns the cells produce collectively. The first step in a cell-based modeling approach is to isolate sub-processes, e.g. the patterning capabilities of one or a few cell types in cell cultures. Cell-based models can then identify the mechanisms responsible for patterning in vitro. This review discusses two cell culture models of morphogenesis that have been studied using this combined experimental-mathematical approach: chondrogenesis (cartilage patterning) and vasculogenesis (de novo blood vessel growth). In both these systems, radically dif- ferent models can equally plausibly explain the in vitro patterns. Quantitative descriptions of cell behavior would help choose between alternative models. We will briefly review the experimental methodology (microfluidics technology and traction force microscopy) used to measure responses of individual cells to their micro-environment, including chemical gradients, physical forces and neighboring cells. We conclude by discussing how to include quantitative cell descriptions into a cell-based model: the Cellular Potts model

Quantitative Limits on Small Molecule Transport via the Electropermeome - Measuring and Modeling Single Nanosecond Perturbations

The detailed molecular mechanisms underlying the permeabilization of cell membranes by pulsed electric fields (electroporation) remain obscure despite decades of investigative effort. To advance beyond descriptive schematics to the development of robust, predictive models, empirical parameters in existing models must be replaced with physics- and biology-based terms anchored in experimental observations. We report here absolute values for the uptake of YO-PRO-1, a small-molecule fluorescent indicator of membrane integrity, into cells after a single electric pulse lasting only 6 ns. We correlate these measured values, based on fluorescence microphotometry of hundreds of individual cells, with a diffusion-based geometric analysis of pore-mediated transport and with molecular simulations of transport across electropores in a phospholipid bilayer. The results challenge the drift and diffusion through a pore model that dominates conventional explanatory schemes for the electroporative transfer of small molecules into cells and point to the necessity for a more complex model

Nonosecond Pulsed Electric Field Induced Changes in Dielectric Properties of Biological Cells

Nanosecond pulsed electric field induced biological effects have been a focus of research interests since the new millennium. Promising biomedical applications, e.g. tumor treatment and wound healing, are emerging based on this principle. Although the exact mechanisms behind the nanosecond pulse-cell interactions are not completely understood yet, it is generally believed that charging along the cell membranes (including intracellular membranes) and formation of membrane pores trigger subsequent biological responses, and the number and quality of pores are responsible for the cell fate. The immediate charging response of a biological cell to a nanosecond pulsed electric field exposure relies on the dielectric properties of its cellular components. Conversely, intense nanosecond pulses will change these properties due to conformational and functional changes. Hence, an understanding of biodielectric phenomena is necessary to explain the underlying interaction mechanisms between nanosecond pulses and biological materials. To this end, we have investigated the changes in dielectric characteristics of biological cells and tissues after exposure to multiple nanosecond pulses. Significant differences have been observed in dielectric properties and membrane integrity of Jurkat cells for exposures to nanosecond and microsecond pulsed electric fields despite delivery of the same energy, suggesting different pore formation and development mechanisms. The effect of nanosecond pulsed electric fields on the dielectric properties of Jurkat cells is long-lasting which is consistent with predictions of much longer pore resealing times for shorter pulses. Strong correlation between short-term plasma membrane conductivity and long-term cell survival has also been observed for different nanosecond-exposure conditions. Together with the studies on tissues, we demonstrate that dielectric spectroscopy is capable of assessing conformational and possibly functional changes of cells after exposure to nanosecond pulsed electric fields on biologically relevant time scales, and in turn, evaluate and compare the efficacy of chosen pulse parameters

Cardiac cell modelling: Observations from the heart of the cardiac physiome project

In this manuscript we review the state of cardiac cell modelling in the context of international initiatives such as the IUPS Physiome and Virtual Physiological Human Projects, which aim to integrate computational models across scales and physics. In particular we focus on the relationship between experimental data and model parameterisation across a range of model types and cellular physiological systems. Finally, in the context of parameter identification and model reuse within the Cardiac Physiome, we suggest some future priority areas for this field