Innovations in Electrostimulation: Expanding Nanosecond Electric Pulse Delivery for Low-Intensity in vivo Therapeutic Applications
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Authors
You, Kyung Eun
Issue Date
2023
Type
Dissertation
Language
Keywords
Chromaffin cell , Nanosecond electric pulse , Radiofrequency
Alternative Title
Abstract
The biological effects of nanosecond electric pulse (NEP) have been investigated, showing that ultrashort pulse stimulation can mimic physiological responses. Our research group has studied NEP-induced cell excitation by using bovine adrenal chromaffin cells, which are served as a neural cell model with characteristics of both the endocrine and nervous systems. The studies demonstrated that a single 5 ns pulse evoked a rise in intracellular calcium levels by opening voltage-gated calcium channels (VGCCs), resulting in catecholamine release.Conventional NEP stimulation methods, while valid primarily for in vitro studies, require improvement to expand their applications, especially for therapeutic purposes. First, an appropriate pulse generation system is required for producing advanced types of electric pulses that contain specific frequency components. Traditionally used pulse generators are not suitable to generate precise waveforms due to a lack of fine control of pulse parameters. Second, the utilization of high intensity may lead to safety concerns in therapeutic applications and restrict the development of battery-operative wearable medical devices. Third, direct current (DC)-based NEP encounters limitations in its ability to reach deeper biological targets due to the high impedance of body tissues. Consequently, the placement of electrodes near the target area is essential to ensure signal delivery, which necessitates invasive approaches.
This study aims to develop novel electrostimulation methods potentially applicable for therapeutic purposes by addressing the limitations of current approaches. To demonstrate the feasibility of our novel pulses, we monitored intracellular calcium level changes in chromaffin cells in response to the pulses generated by a direct digital synthesizer (DDS)-based NEP generation system. DDS provides flexibility and precision in pulse shaping as it enables by the generation of arbitrary waveform signals based on user-provided waveforms. However, commercial DDSs are constrained by an upper voltage limit of 10 V, which fails to satisfy the voltage needed for cell stimulation in this study. Hence, a custom-built DDS system, composed of four commercial DDS in series, was designed and employed. However, the resulting system is confined by its limited voltage output (< 40 V) and encounters substantial signal attenuation at higher frequencies, primarily due to unavoidable imperfect impedance matching. As a result, the range of frequency components in this study was curtailed. This limitation will be addressed in future work through the redesign and optimization of the next-generation DDS system, thereby expanding its capability.
In this study, three novel electrostimulation pulses have been developed and characterized by time and frequency analysis. In addition, the experimental data with chromaffin cells supported their validity to evoke cell responses and demonstrated the impact of pulse parameters on the stimulations.
A low pass filtered (LPFed) 5 ns pulse was specifically designed to maintain the advantages of a 5 ns pulse, leading to transient Ca2+ responses by preserving the key pulse parameters while broadening the pulse width to achieve a low-intensity pulse. Although the pulse shape was successfully obtained, our pulse generation system has a significant voltage drop with increasing frequency, resulting in an unsuccessful experimental validation of this novel pulse.
To enhance penetration depth, we developed radiofrequency (RF)-based pulses of multitone RF pulses and chirps. These pulses were synthesized by combining individual AC signals to produce a signal that is effective for cell stimulation. The multitone RF pulses have three components of AC pulses that have different frequencies. The two high-frequency RF signals are in the MHz level and the relatively low-frequency pulse piggybacks the high-frequency pulses to shift toward the positive phase, which improves the cell membrane charging effects. In this pulse stimulation, a reduction in pulse intensity was achieved by extending pulse width and adopting the concept of MHz compression. For multitone RF pulses, 126 and 245 ns of average pulse widths were examined by giving 5-100 cycles at up to 4 MHz of repetition rates, which resulted in an 18.5-fold reduced E-field compared to the 5 ns pulse. The multitone RF pulses evoke cell responses in chromaffin cells, and their effectiveness was notably influenced by the frequency and voltages of component pulses. Certain pulse conditions, however, could lead to enhanced cell response outcomes compared to the stimulation with DC offset.
Chirp is an RF signal whose frequency is continuously changing in time. In this study, we used a non-linear chirp to increase a positive portion of the signal. To optimize the frequency response for enhancing the cell membrane charging effect, we combined two non-linear chirps that have opposite frequency-changing patterns, to form a composite chirp. The composite chirp has more intensive low-frequency components compared to non-linear chirps, consequently enhancing its cell stimulation effect. The range of pulse widths used in chirp stimulations was 146.3- 360 ns and up to 1000 cycles were delivered to the chromaffin cells, which reduced the E-field amplitude required for cell stimulations. The lowest E-field that could evoke cell responses with reliable cell response rates (>75%) was 1.13 kV/cm, which was 2.4 times smaller than the E-field used in the multitone RF pulses and 44.2 times smaller than that of the 5 ns pulse. In addition, the composite chirps with relatively low E-field and smaller pulse widths tend to produce transient cell responses despite decreasing the cell response rates.
To achieve non-invasive stimulation, the signals are desired not to affect the cells in the pathway of delivery but stimulate the target cells. Therefore, our RF pulses are designed to consider that the component pulses are individually emitted from different surface locations of in vivo subjects and then combined at target sites. This study experimentally demonstrated the potential as the component pulses were less effective for evoking Ca2+ influx in chromaffin cells compared to the combined pulses at certain pulse conditions. However, the results were dependent on the pulse parameters, which interact with each other to change the cell response. Therefore, the exact pulse parameters which can maximize both advantages of delivery of signals and stimulating cellular responses need to be investigated with more detailed pulse parameters.
In summary, this dissertation lays the groundwork for the development of advanced electrostimulation methods that are potentially applied for therapeutic purposes. The study suggested approaches to overcome the current challenges to comply with regulations of medical applications and demonstrated the validity of the pulses. However, further development is required, with refining pulse shapes and specified pulse parameters to identify critical points that affect cell response patterns. Furthermore, in vivo studies on non-invasive signal delivery are necessary to realize the full potential of our novel pulses.