Subcellular electrical stimulation of neurons enhances the myelination of axons by oligodendrocytes

<div><p>Myelin formation has been identified as a modulator of neural plasticity. New tools are required to investigate the mechanisms by which environmental inputs and neural activity regulate myelination patterns. In this study, we demonstrate a microfluidic compartmentalized culture system with integrated electrical stimulation capabilities that can induce neural activity by whole cell and focal stimulation. A set of electric field simulations was performed to confirm spatial restriction of the electrical input in the compartmentalized culture system. We further demonstrate that electrode localization is a key consideration for generating uniform the stimulation of neuron and oligodendrocytes within the compartments. Using three configurations of the electrodes we tested the effects of subcellular activation of neural activity on distal axon myelination with oligodendrocytes. We further investigated if oligodendrocytes have to be exposed to the electrical field to induce axon myelination. An isolated stimulation of cell bodies and proximal axons had the same effect as an isolated stimulation of distal axons co-cultured with oligodendrocytes, and the two modes had a non-different result than whole cell stimulation. Our platform enabled the demonstration that electrical stimulation enhances oligodendrocyte maturation and myelin formation independent of the input localization and oligodendrocyte exposure to the electrical field.</p></div

Whole cell and subcellular ESTIM enhances oligodendrocyte precursor cells (OPCs) differentiation into oligodendrocytes (OLs) and supports axon myelination.

<p>(A-D) Representative images of axons in the axonal compartment stained against neurofilaments (NF, red) and OLs stained against a set of differentiation markers: (A) O4 signal (green) expressed by premature OPCs (white arrows) after 3 days of stimulation; (B) CNPase signal (green) expressed by mature OLs (white arrows) after 7 days of stimulation; (C) MBP signal (green) expressed by mature OLs (white arrows) after 7 days of stimulation; (D) MBP signal (green) expressed by mature OLs after 14 day of stimulation demonstrating formed myelin fragments (white arrows). Scale bar (A—D): 50μm. The images for all experimental conditions are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0179642#pone.0179642.s006" target="_blank">S6 Fig</a>. (E) The percentage of O4-positive OPCs decreased slightly after 3 days in the control group outlining the baseline level of the differentiation. ESTIM further decreased the percentage of O4-positive OPCs. (F-G) 7 days of ESTIM supported OLs maturation as indicated by the increase in the percentage of (F) MBP-positive and (G) CNPase-positive cells. (H) 14 days of ESTIM increased the number of formed myelin fragments. The data are presented as mean ± S.E.M. For individual data points see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0179642#pone.0179642.s007" target="_blank">S7 Fig</a>. The numbers on the graphs stand for the numbers of experimental replicates. The groups were compared with one-way ANOVA at a significance level of α = 0.05; *** p<0.001.</p

Electric field strength within the long compartments.

<p>(A) Schematic of the simulation model with larger compartment length (15 mm). (B) Cross-section of model along indicated plane. (C-E) Contour maps of electric field magnitudes within the compartments for different electrode configurations.</p

Integrated compartmentalized and electrical system for subcellular stimulation of neurons.

<p>(A) Dorsal root ganglion neurons and oligodendrocyte precursor cells were cultured in microfluidics and electrically stimulated in three different modes of electrode configuration: whole cell stimulation (WholeSTIM), somatic compartment stimulation (SomaSTIM) and axonal compartment stimulation (AxonSTIM). (B) Schematic of stimulation protocol.</p

Electrode configuration affects the effectiveness of neuron stimulation.

<p>(A) The same central regions of a microfluidic neuron culture (broken line boxes) were first imaged under electrode configuration D than electrode configuration C. (B) DIC images of the representative region of the imaged areas. (C-D) Calcium response of cell bodies (<i>C</i>) and axons (<i>D</i>) to ESTIM. E Average calcium changes (ΔF/F_B) pooled from the same cell bodies in two different electrode configurations. The inset shows frequency of the firing events (f_ΔF/FB) in 3 min following ESTIM onset. The results are expressed as mean ± SEM (n = 10 cells). Statistical analysis was performed with paired student-t test ** p<0.005.</p

Electric field amplitude within the compartments.

<p>(A) Schematic of the simulation model. (B) Cross-section of model along indicated plane. (C-H) Contour map of electric field amplitude within compartments for different stimulation electrode configurations. (I-K) Profile of electric field amplitude along dashed lines in (C-H) spanning two wells. (L) Measured (gray; relative electrical potential difference) and simulated (black; relative electric magnetic field) at 6 locations, as numbered in (<i>C</i>) and (<i>E</i>) and outlined (white x and dotted lines) in all electrodes configurations (<i>C-H</i>). The data for G and H come from the same set of simulations and measurements. Measurement data are mean ± S.D. For individual data points see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0179642#pone.0179642.s002" target="_blank">S2 Fig</a>.</p

High-performance wireless powering for peripheral nerve neuromodulation systems

<div><p>Neuromodulation of peripheral nerves with bioelectronic devices is a promising approach for treating a wide range of disorders. Wireless powering could enable long-term operation of these devices, but achieving high performance for miniaturized and deeply placed devices remains a technological challenge. We report the miniaturized integration of a wireless powering system in soft neuromodulation device (15 mm length, 2.7 mm diameter) and demonstrate high performance (about 10%) during <i>in vivo</i> wireless stimulation of the vagus nerve in a porcine animal model. The increased performance is enabled by the generation of a focused and circularly polarized field that enhances efficiency and provides immunity to polarization misalignment. These performance characteristics establish the clinical potential of wireless powering for emerging therapies based on neuromodulation.</p></div

Conformal wireless powering transmitter.

<p>A: Transmitter consisting of concentric metal rings. Adjacent rings are connected and loaded with reactive elements along the <i>x</i> and <i>y</i> axes to generate circularly polarized (CP) field or linearly polarized (LP) field. B: Simulated power transfer efficiency to a 20-mm straight dipole as a function of its orientation. The transmitter is placed above a tissue medium. The dipole is 10 mm deep in tissue. C: Contour plot of the electric field intensity generated by reactive elements along the <i>x</i> axis. D: Photograph of the transmitter. E: Numerically simulated and measured power transfer efficiency to a 20-mm long dipole as a function of its depth in tissue. F: Numerically simulated and measured power transfer efficiency over a curved surface. G: Simulated specific absorption ratio (SAR) using a human voxel model. Measured efficiency is recorded in saline solution. Simulations and measurements are performed at 2.4 GHz.</p

Performance and electrical properties of wireless cuff electrodes.

<p>A—C: Simulated performance of a 20-mm long straight dipole versus a 14-mm meandered dipole as a function of operating frequency. The dipoles are placed 10 mm deep in a tissue medium. D: Efficiency of the integrated circuits in converting radio-frequency power into current across loading impedances of 500 Ω and 1000 Ω (<i>n</i> = 2, minimum, mean, maximum). E: Real and imaginary parts of electrode impedance spectrum recorded in saline solution. F: Electrode impedance at 1 kHz after applying pulse train. Pulse train parameters, 25 Hz, 1 V amplitude, 500 <i>μ</i>s pulse width.</p

Real-time <i>in vivo</i> responses.

<p>First row records ECG waveform from lead III. Second row shows spikes extracted from the ECG waveform. Third row shows heart rate oscillation during 10-s alternating on-off stimulation. Forth row records blood pressure waveform. Fifth and sixth rows show the decrease in systolic and diastolic pressures during the 10-s alternating on-off stimulation.</p