Effect of Pulse Interval on Promoting Secretion of Neurite-Inducing Factor from Glioma Cells in the Application of Nanosecond High Electric Field Pulses
Authors: Mori Chisato , Shinohara Hiroaki , Suga Minoru 
Published: December 2020

1. Purpose

We previously discovered and reported that the application of nanosecond high electric field pulses transiently increased intracellular Ca2+ concentration in glioma cells to promote the secretion of neurite-inducing factor. Moreover, it has been clarified that the electric field strength, the number of pulse application, and the pulse width have a strong effect on the secretory ability as a condition for promoting the secretion of these neurite-inducing factors. In this presentation, we further report that even nanosecond pulses with a weak electric field strength which does not cause the increase of intracellular Ca2+ concentration with a single pulse can induce the intracellular Ca2+ increase and neurite-inducing factor secretion from glioma cells by applying multiple pulses with short intervals. The considered mechanism also will be discussed in this presentation.

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2. Experimental method

Rat glioma C6 cells were seeded on an ITO electrode and cultured overnight to adhere. To monitor intracellular Ca2+ concentration the cells were treated with a medium containing Fura-2 AM for 30 minutes to introduce the Ca2+ indicator into the cells. After washing the cells on the electrode, Hanks Balanced Salt Solution (HBSS) was put in the chamber, and an aluminum plate electrode serving as a counter electrode was faced to ITO electrode with a spacer in thickness of 30 µm, and set on the stage of an inverted fluorescence microscope. After two minutes from the start of measurement, nanosecond electric field pulse was applied in various voltage and pulse interval, and changes in intracellular Ca2+ concentration were observed. Furthermore, thapsigargin and 2-aminoethoxydiphenylborane (2-APB) were used to confirm that the increase in intracellular Ca2+ concentration may be due to Ca2+ release from the endoplasmic reticulum via IP3 receptors. In addition, after applying a nanosecond high electric field pulse to C6 cells in the same manner in a clean bench, the HBSS was immediately replaced with DMEM medium containing 10% FBS, and the cells were cultured for 1 day or 2 days. The culture supernatant (Conditioned Medium: CM) of the C6 cells was then collected, and exchanged completely with the medium of PC12 cells, which is a neural progenitor cell model. The morphological change of PC12 cells was observed and recorded for 6 days. The PC12 cells which extended the neurites more than twice as the cell body were recognized as neuronal differentiated cells. By calculating the differentiation rate of PC12 cells, it was evaluated how the secretion of the neurite-inducing factor from C6 cells was promoted or not.

3. Results and discussion

Three pulses with a pulse width of 140 ns and an electric field strength of 23 kV / cm were applied at intervals of 2 seconds, 30 seconds, and 1 minute to C6 cells and intracellular Ca2+ concentration changes and neurite-inducing factor secretion was examined. It was demonstrated that intracellular Ca2+ gradually increased at shorter intervals of 2 seconds and 30 seconds, but not increased at intervals of 1 minute. In addition, intracellular Ca2+ concentration increase was observed even when pulse application was done in the absence of extracellular Ca2+. It indicated that this increase in intracellular Ca2+ concentration is due to Ca2+ release from organelles. Furthermore, intracellular Ca2+ concentration elevation was not induced by the same pulse application after treatment with thapsigargin or 2-APB. Therefore, it was considered that Ca2+ was released from the endoplasmic reticulum via IP3 receptors by the pulse application in this condition. Figure 1 (a) and (b) show images of PC12 cells cultivated in the CM of C6 cells which were exposed to three pulses with 1 minute or 2 seconds intervals, respectively. From these results, it was demonstrated that the secretion of the neurite-inducing factor was promoted by the multi-pulse application with short intervals. Furthermore, it was found that the Ca2 + release via IP3 receptors of the endoplasmic reticulum and following promotion of the secretion of neurite-inducing factor could be induced by shortening the nanosecond multi-pulse interval in the application of weak electric field. Now, we are planning to clarify what kind of gene expression in C6 cells is induced by pulse application to identify and quantify the secreted neurite-inducing factor.

Figure 1