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

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 ([Ca²⁺]_i) based on ES intensity and duration. In this article, we validate the computation model for the [Ca²⁺]_i changes in neuron soma. Embryonic chicken dorsal root ganglion cells were suspended in 3-dimensional collagen scaffolds. Fura-2 was used to measure [Ca²⁺]_i 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 [Ca²⁺]_i elevation for ES pulses 100 µs or greater in duration. Nickel (200 µM) and cadmium (200 µM) blocked 98-99% of the [Ca²⁺]_i rise, indicating that the rise in [Ca²⁺]_i in response to ES is via voltage-dependent calcium channels. The average [Ca²⁺]_i rise in response to ES was about one-tenth of the peak rise. Therefore, the computational model is validated for elevating [Ca²⁺]_i of neurons and can be used as a tool for designing efficacious ES protocols for improving neuronal regeneration.NEW & NOTEWORTHY Electrical stimulation is used to enhance neuron growth, and the role of neuronal intracellular calcium concentration ([Ca²⁺]_i) 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 [Ca²⁺]_i in response to a broad and defined range of electrical pulse durations and intensities. These results validate our previously published novel computational model of [Ca²⁺]_i.