Validation of electrical stimulation models: intracellular calcium measurement in three-dimensional scaffolds

Abstract

Peripheral nerve injury can be disabling. Regeneration is limited by the rate of axonal extension, and proximal injury to peripheral nerves can take over a year to reach target organs. Electrical stimulation (ES) has been shown to increase the rate of neurite growth, though the mechanism is not yet well understood. In our prior manuscript, we developed a computational model that demonstrates how ES can functionally elevate intracellular calcium concentration ([Ca]) based on ES intensity and duration. In this article, we validate the computation model for the [Ca] changes in neuron soma. Embryonic chicken dorsal root ganglion cells were suspended in 3-dimensional collagen scaffolds. Fura-2 was used to measure [Ca] in response to biphasic ES pulses ranging from 70 to 60,000 V/m in intensity and from 10 µs to 100 ms in duration. The computational model most closely matched the experimental data of the neurons with the highest [Ca] elevation for ES pulses 100 µs or greater in duration. Nickel (200 µM) and cadmium (200 µM) blocked 98-99% of the [Ca] rise, indicating that the rise in [Ca] in response to ES is via voltage-dependent calcium channels. The average [Ca] rise in response to ES was about one-tenth of the peak rise. Therefore, the computational model is validated for elevating [Ca] of neurons and can be used as a tool for designing efficacious ES protocols for improving neuronal regeneration. Electrical stimulation is used to enhance neuron growth, and the role of neuronal intracellular calcium concentration ([Ca]) is an area of research interest. Widely varying stimulation parameters in the literature make it difficult to compare stimulation protocols. The results in this manuscript are the first to show neuronal [Ca] in response to a broad and defined range of electrical pulse durations and intensities. These results validate our previously published novel computational model of [Ca].

Location Specificity of Transcranial Electrical Stimulation on Neuronal Electrodynamics: A Mathematical Model of Ion Channel Gating Dynamics and Ionic Flux Due to Neurostimulation

“…These results are consistent with prior experiments that found an increase in calcium concentration due to an influx of calcium in the presence of electrical stimulation (Islam et al, 1995; Adams et al, 2017). In fact, the values predicted by the model are within one order of magnitude of those shown in electrical stimulation biological studies (Adams et al, 2017). Moreover, the model augments this knowledge by provding a detailed prediction of how, where, and when calcium ion flow into the neuron as described in section 3.3.…”

“…These results are consistent with published TES studies that show an increase in calcium influx from a membrane depolarization due to an electric field applied in the extracellular medium (Nitsche et al, 2003; Adams et al, 2017). In addition, like the biological literature, our model predicts that this influx is governed by voltage gated calcium channel permeability (Islam et al, 1995).…”

“…Further, TES has shown to improve language re-learning in dementia patients (Wang et al, 2013; Yun et al, 2016). In addition to clinical findings, biological experiments have begun to show the effects of TES on membrane polarization (Liebetanz et al, 2002; Bikson et al, 2004; Stagg and Nitsche, 2011; Das et al, 2016) and calcium homeostasis (Islam et al, 1995; Nitsche et al, 2003; Adams et al, 2017), however difficulties in capturing ion channel state, ionic flux, and intracellular calcium concentrations continuously over time with a high sampling frequency yields limited neurostimulation data at the cellular level (Adams et al, 2017). Thus, the direct influence of an applied TES electric current on voltage-gated ion channel states as well as other cellular level mechanism by which TES operates is largely unknown (Nitsche et al, 2008).…”

Noninvasive Stimulation of Neurotypic Cells Using Persistent Photoconductivity of Gallium Nitride

“…However, an overwhelming amount of data in the literature have established that an increase in the intracellular calcium concentration is one of the steps of electrical stimulation of neurotypic cells. 24 In all of our experiments, the cells are never exposed to UV light. The n-type Ga-polar GaN is exposed to UV light before any introduction into the cell culture dishes.…”

Variably doped nanostructured gallium nitride surfaces can serve as biointerfaces for neurotypic PC12 cells and alter their behavior

“…26 In order to quantify this communication, Fluo-4, a commonly-used dye to quantify [Ca 2+ ] i was used. 27,28 Previous work only quantied the immediate non-invasive cell stimulation. Though the concept was proven, expanding the applicability of these PPC effects requires greater understanding of materials' functionality.…”