Characterization of Schwann cells stimulated by DC electric fields

Abstract

Schwann cells (SCs) are PNS glia with numerous neuron-supporting functions, including myelination of axons. Although lesser discussed, SCs also fulfill many important roles after peripheral nerve injury (PNI) contributing significantly to the PNS regeneration process. Clusters of congregated SCs (Bands of Bungner) precede axon regeneration and facilitate the growth of extending axons to their distal targets which is particularly important in the lesion area of severed nerves. While this phenomenon occurs naturally, recovery from PNI can still be inadequate, especially in nerve transection or large gap injuries. Current treatments for nerve transection injuries are limited to coaptation of the nerve via sutures or nerve grafts. However, poor functional outcomes or donor site morbidity remain unaddressed problems. At the cellular level, axon pathfinding and extension relies heavily on the interaction between SCs and axonal growth cones. Depletion or removal of SCs at the lesion has been implicated to poor functional outcomes. With their pivotal role throughout nerve regeneration, we theorize axon regeneration can be improved by augmenting the SC population at the site of injury by encouraging migration to the lesion and via expression of morphological phenotypes that imitate the Bands of Bungner. DC electric fields (EFs) have been well studied in the past as a method to modulate cell orientation and migration and within the context of the nervous system, have been used to promote regeneration in lesioned spinal cords. However, very little work has investigated the effects of electrical stimulation on glia, such as SCs. Existing literature is lacking with regards to various aspects of SC responses, including direction of alignment. We hypothesize electrical stimulation can modulate SC behavior to reinforce/replicate behaviors observed within Bands of Bungner, which may be developed into a treatment for victims suffering peripheral nerve injury. We begin the current study with a thorough investigation into electric field modulated SC behavior. Using conventional 2D cell culture we demonstrate SC sensitivity to EFs by analyzing alignment, morphology and migration data. We employed EFs within the physiologic range. Waveforms used were constant DC as well as a 50% duty cycle DC and an oscillating DC. The latter two may prove more appropriate in vivo due to reduced accumulation of cytotoxic byproducts generated at the electrode interfaces. Our results highlight the sensitivity of SCs to DC electric fields of varying waveforms. SCs showed a strong propensity to align perpendicular to the field and display some cathodal migration in 2D cultures. Additional studies with variable cell density revealed cell-cell interaction further enhanced the alignment response. To more closely replicate the nerve microenvironment, a 3D cell culture model of PNI was created. Embedded in matrices, we found SCs displayed weaker migratory and alignment responses compared to 2D results. The direction of galvanotaxis was reversed, with SCs migrating toward the anode. Both alignment and migratory responses have potential applications for PNI. The galvanotactic behavior of SCs could be used to boost the SC population, increasing the number of Bands of Bungner. Cell alignment would be particularly advantageous at the lesion where axon regeneration is most difficult without the physical guidance of endoneurial tubes. This study characterizes SC behavior in applied EFs using conventional 2D and 3D cell culture techniques. We found SCs are sensitive to electric stimulation, supporting the idea that applied EFs could be used to indirectly promote regeneration in damaged peripheral nerve by modulating SC response after injury. Potential applications include generating an EF across damaged nerves to align SCs, especially in the lesioned area, using EFs to induce SC migration to the lesion to increase the number of cells guiding severed axons, and pre-aligning SCs in synthetic nerve grafts.