Validation of Electrical Stimulation Models: Intracellular Calcium Measurement in 3-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 ([Ca2+]i) based on ES intensity and duration. Here, we validate the computation model for the intracellular [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 the concentration of [Ca2+]i in response to biphasic ES pulses ranging from 70 V/m to 60,000 V/m in intensity, and 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.

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