Electrode stimulation parameter modifications elicit differential glial cell responses in vitro over a short 4-h timecourse

Abstract

A cell culture model to assess glial cell responses to electrically stimulating electrodes in real-time was developed. Our previous work measured glial cell responses to stimulation paradigms and highlighted the importance of electrical stimulation considerations when designing a biocompatible neural interfacing device. The formation of voids around stimulating platinum-iridium electrodes also prompted an investigation into the fate of cells that would have once populated that area. Live-imaging experiments involving EGFP-positive microglia from heterozygous CX3CR-1^+/EGFP mice were designed. Live-imaging animations over 4 h showed necrotic microglial cell death around stimulating electrodes. The degree to which this was occurring was further analyzed by electrically stimulating mixed glia and modifying parameters such as stimulation amplitude (0.1–0.4 mA), waveform shape (rectangular/sinusoidal/ramped), and frequency (25–55 Hz). The different stimulation parameters had differential effects on glial cell biomarker signal outputs (cell density, fluorescence intensity, area coverage). Scanning electron microscopy and energy-dispersive x-ray spectroscopy of the electrode surfaces post-stimulation did not reveal any significant damage or changes to surface elemental composition. Finally, electrochemical testing of the proposed in vitro setup revealed influences of different components of the mixed glial cell cultures towards the electrochemical performance of the electrodes in terms of cathodic charge storage capacity, impedance, phase angle, and voltage transient excursions. The results highlight the impact that electrical stimulation parameters have on glial cell fate at the electrode-cell culture interface, and provide data towards refinement of stimulation paradigms used in electrical neuromodulation applications.