Biphasic monopolar electrical stimulation induces rapid and directed galvanotaxis in adult subependymal neural precursors

Abstract

IntroductionFollowing injury such as stroke, adult mammalian subependymal neural precursor cells (NPCs) are induced to proliferate and migrate toward the lesion site where they differentiate into neural cells, albeit with limited efficacy. We are interested in enhancing this migratory ability of NPCs with the long-term goal of promoting neural repair. Herein we build on our previous studies demonstrating that direct current electric fields (DCEFs) promote rapid and cathode-directed migration of undifferentiated adult NPCs (but not differentiated phenotypes) - a phenomenon known as galvanotaxis. While galvanotaxis represents a promising strategy to promote NPC recruitment to lesion sites, stimulation of neural tissue with DCEFs is not a clinically-viable strategy due to the associated accumulation of charge and toxic byproducts. Balanced biphasic waveforms prevent the accumulation of charge and thus are outside of the limitations of DCEFs. In this study, we investigated the effects of balanced biphasic electrical stimulation on the migratory behaviour of undifferentiated subependymal NPCs and their differentiated progeny.MethodsNPCs were isolated from the subependymal zone of adult mouse brains and cultured in a NPC colony-forming assay to form neurospheres. Neurospheres were plated onto galvanotaxis chambers in conditions that either promoted maintenance in an undifferentiated state or promoted differentiation into mature phenotypes. Chambers containing cells were then time-lapse imaged in the presence of either biphasic monopolar, or biphasic bipolar electrical stimulation, or in the complete absence of electrical stimulation. Single cell migration was subsequently tracked and the cells’ magnitude of velocity, directedness and tortuosity were quantified.ResultsWe demonstrate, for the first time, the use of balanced biphasic electric fields to induce galvanotaxis of NPCs. Undifferentiated adult mouse subependymal NPCs exposed to biphasic monopolar stimulation undergo rapid and directed migration toward the cathode. In contrast, both biphasic bipolar stimulation and the lack of electrical stimulation produced non-directed migration of NPCs. Notably, NPCs induced to differentiate into mature phenotypes prior to exposure to electrical stimulation do not migrate in the presence or absence of biphasic stimulation.ConclusionWe purport that balanced biphasic stimulation represents a clinically-viable technique for mobilizing NPCs that may be integrated into strategies for promoting endogenous neurorepair.Electronic supplementary materialThe online version of this article (doi:10.1186/s13287-015-0049-6) contains supplementary material, which is available to authorized users.

Glial responses to implanted electrodes in the brain

“…In the context of neurogenesis, neuromodulation is gaining traction as a reparative tool for brain injury and disease 118,134 . Neurostimulation stimulates neural progenitor proliferation 135–137 , directs the migration (galvanotaxis) of neuronal and glial precursors 134,138–140 and promotes their differentiation 136,137,140,141 . Interestingly, tDCS-polarized proinflammatory microglia accompany NG2-precursor migration to promote functional recovery after stroke 130 .…”

A 3D Printed Device for Low Cost Neural Stimulation in Mice

“…Mice were anesthetized with 1.5–2.5% isofluorane, placed in the stereotaxic and the implanted electrode was interfaced with a biphasic electrical stimulator. Mice received stimulation for 1 h using pulse parameters: 1–200 μA amplitude, 50–500 μs pulse width, 1–285 Hz pulse repetition frequency, as previously described by Babona-Pilipos et al (2015). Following each stimulation session, mice were returned to their home cages for 24 or 72 h. Mice were sacrificed with an overdose of Avertin, transcardially perfused with 4% ice-cold paraformaldehyde and the brains removed.…”

Electrotaxis of Glioblastoma and Medulloblastoma Spheroidal Aggregates

“…Babona-Pilipos, et al . studied neurospheres of subependymal neural precursors plated on a 2D substrate, but only analysed cells that had migrated sufficiently far from the rest of the neurosphere cohort 27 . Cohen, et al ., developed methods to track individual cells in epithelial sheets of arbitrary geometries, as a way to piece apart the varied electrotactic responses of leader and follower cells 28 .…”

“…Zhao, et al ., developed a clever, high-throughput method of assaying cells in arrays of 3D agarose droplets with Dictyostelium cells, though this method has yet to be attempted for mammalian cell culture 31 . Spheroids have also been used as a 3D format though the studies have not included a 3D substrate and the spheroids have not been assessed for collective migration 27,32 . Most recently, the above-mentioned study by Huang, et al ., on brain-tumor initiating cells that had differing electrotactic responses in 3D and 2D, highlights the need for more studies that consider experiments more than just 2D, single cell assays 25 .…”