3D axon growth by exogenous electrical stimulus and soluble factors

Axon growth and alignment are fundamental processes during nervous system development and neural regeneration after injury. The present study investigates the effects of exogenous stimulus of electrical signals and soluble factors on axon 3D growth, using a silk protein material-based 3D brain tissue model. Electrical stimulus was delivered via embedded gold wires positioned at the interface of the scaffold region and the center matrix gel-filled region, spanning the axon growth area. This setup delivered applied electrical field directly to growing axons, and the effects were compared to micro-needle assisted local delivery of soluble factors of extracellular (ECM) components and neurotrophins. Dissociated rat cortical neurons were exposed to an alternating field of 80 mV/mm at 0.5 Hz to 2 kHz or soluble factors for up to 4 days, and evaluated by of β III-tubulin immunostaining, confocal imaging and 3D neurite tracing. 0.5-20 Hz were found to promote axon growth, with 2 Hz producing the biggest effect of ∼30% axon length increase compared to control cultures. Delivery of ECM components of laminin and fibronectin resulted significantly greater axon initial length increases compared to neurotrophic factors, such as BDNF, GDNF, NGF and NT3 (all at 1 μM). Though axon lengths under 2 Hz stimulation and LN or FN exposure were statistically similar, significant AC-induced axon alignment was found under all frequencies tested. The effects included perpendicular orientation of axons trespassing an electrode, large populations of aligned axon tracts in parallel to the field direction with a few perpendicularly aligned along the middle point of the EF. These findings are consistent with the hypothesis that an electrode in AC field could act as an alternating cathode that attracts the growing tip of the axon. These results demonstrate the use of alternating electric field stimulation to direct axon 3D length growth and orientation. Our study provides basis for further optimizing stimulation parameters, in conjunction of delivery of growth promoting soluble factors to direct axon growth in a brain mimetic 3D environment. This system provides a platform for studying the effects of exogenous signals on nervous system development and for testing neuromodulation approaches for neurological diseases.