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Electromagnetic Analysis of Exposure Systems – Potential Factors Underlying the Variation in Duration of Nanosecond Electric Pulse-Evoked Changes in Intracellular Calcium Level in Adrenal Chromaffin Cells
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Studying the effects of the nanosecond electric pulses (NEPs) on excitable cells, especially on bovine adrenal chromaffin cells, has been of great interest to our research group. Chromaffin cells undergo a process of exocytosis to release catecholamines from their secretory granules. Exocytosis is triggered when the cells are stimulated with the physiological stimulus that activates acetylcholine nicotinic receptor. This activation leads to an influx of Ca2+ into the cell, which can be imaged using the fluorescence microscopy technique. Imaging reveals that in response to the application of physiological stimulus the cells undergo an initial rapid increase in intracellular calcium concentration ([Ca2+]i) followed by a decrease, resulting in a short-lived Ca2+ response. Similar to nicotinic receptor activation, a single 5 ns electric pulse triggers a rapid increase in [Ca2+]i. However, the return of [Ca2+]i to pre-stimulus level is slower than that triggered by the physiological stimulus. In addition, it was found that the duration of NEP-induced Ca2+ responses varied according to the manner in which the cells were exposed to the 5 ns pulse. In our research, the first NEP delivery system that was used consisted of a gold strip chamber. Cells exposed to a 5 ns, 5 MV/m pulse with this exposure system resulted in short-lived (< 10 s) Ca2+ responses, similar to that evoked by physiological stimulus (5 - 6 s). However, when chromaffin cells were stimulated by a 5 ns, 5 MV/m pulse using a pointy rod electrode exposure system, Ca2+ responses were more prolonged, in some cases such that [Ca2+]i never returned to pre-stimulus levels during the monitoring period. Because the basis of this prolonged rise in [Ca2+]i is not yet known, the main objective of this project is to understand the potential reasons for the variable duration and prolonged increase in [Ca2+]i by the pointy rod electrode exposure system. The starting premise is that specific electrical and/or the geometrical configuration of the exposure systems are factors. Because NEPs (the source for the exposure systems) are electromagnetic waves, analytical and 3D computational electromagnetics studies of the two exposure systems were performed, and the detailed interactions of the electric field (E-field) with cells in each exposure chamber were analyzed. The first objective was to analyze the time and frequency responses of the pointy rod electrode using the Finite Difference Time Domain (FDTD) method. This was necessary to determine if the geometry of the pointy rod electrode acts as a high- or low-pass filter that could distort the NEP signal at the location of the cell and induce a different type of stimulus than that induced by the gold strip chamber. The simulation results confirmed that the pointy rod electrode delivers the original pulse shape to the location of the cell without distortion. We also carried out simulations and measurements in which we determined that factors related to the fabrication of the pointy rod electrodes (for example, slightly different electrode lengths and types of resistors) also do not significantly distort the pulse shape and frequency spectrum at the cell location. The next major consideration was related to the E-field propagation aspect in the two exposure systems. For the pointy rod electrodes, NEP reflection may occur on the surface of the glass cell dish where the cells are attached, and the scattered NEP can re-radiate to the cell, leading to a higher stimulatory effect of the NEP. To access this possibility, an electrically equivalent round cell, referred to as a spherical capacitor model (SCM), was created in the FDTD software and used to compute the E-field distribution at the cell location. The results indicated that with the pointy rod electrode model the reflected E-field caused a high E-field at the bottom membrane of SCM. In contrast, there was no reflection of the NEP for the gold strip chamber. This result may explain the reason for the longer duration of Ca2+ responses in cells exposed to a NEP using the pointy rod electrode. That is, the cells are exposed to the initial E-field then re-exposed by the reflected E-field, which results in stimulation of the cells with a higher E-field. The E-field distribution of the pointy rod electrode obtained from the numerical modeling revealed an unexpected, localized E-field spike that occurs between the cell membrane and the glass surface on which the cell is attached in the dish. That is, the E-field is concentrated in the narrow gap between the round cell membrane and the flat glass dish, a situation that is not present in the gold strip chamber where cells are in suspension and not attached to a glass surface. This E-field increase at the bottom of the cell can be another source of a higher E-field to which a cell is exposed. In addition, the level of attachment of individual cells to the glass surface can vary such that the cell membrane is flattened to different extents. This results in cells having different elliptical shapes due to the level of attachment to the glass surface. To replicate a realistic cell geometry for this condition in an FDTD model, an elliptical capacitor model (ECM) was modeled, and the resulting E-field distribution was compared with that of the spherical cells (SCM). The results showed that there is a larger area of a localized high E-field in the ECM than in the SCM, indicating that the shape of the cells contributes to the variation of the E-field in the cell membrane. Since attached cells will have different shapes, this could explain why the Ca2+ responses vary even for cells in the same dish. Based on the numerical modeling results described above, several electrodes intended to reduce the E-field reflections were designed, fabricated and tested in Ca2+ imaging experiments. These included a coupler shaped electrode, a flat copper-based electrode and a series of pointy rod electrodes with various incident angles (20°, 45°, and 70°). Unfortunately, the new electrode models did not achieve the shorter duration Ca2+ responses in chromaffin cells observed in cells stimulated with 5 ns pulse in the gold strip chambers. In summary, this study identified multiple factors that could underlie the variations in Ca2+ responses in chromaffin cells evoked by pointy rod electrode exposure system that are not associated with the gold strip chamber exposure system. The study also eliminated some potential factors, such as inconsistencies in electrode fabrication and the selection of electrical components, as contributing to Ca2+ variability. Future work of the project includes investigating the effect of 5 ns pulses with different rise times as the gold strip chamber and pointy rod electrodes were fed by 5 ns pulses with different rise and fall times.