Laminar stream of detergents for subcellular neurite damage in a microfluidic device: A simple tool for the study of neuroregeneration

Abstract

Objective. The regeneration and repair of damaged neuronal networks is a difficult process to study in vivo, leading to the development of multiple in vitro models and techniques for studying nerve injury. Here we describe an approach for generating a well-defined subcellular neurite injury in a microfluidic device. Approach. A defined laminar stream of sodium dodecyl sulfate (SDS) was used to damage selected portions of neurites of individual neurons. The somata and neurites unaffected by the SDS stream remained viable, thereby enabling the study of neuronal regeneration. Main results. By using well-characterized neurons from Aplysia californica cultured in vitro, we demonstrate that our approach is useful in creating neurite damage, investigating neurotrophic factors, and monitoring somata migration during regeneration. Supplementing the culture medium with acetylcholinesterase (AChE) or Aplysia hemolymph facilitated the regeneration of the peptidergic Aplysia neurons within 72 h, with longer (p < 0.05) and more branched (p < 0.05) neurites than in the control medium. After the neurons were transected, their somata migrated; intriguingly, for the control cultures, the migration direction was always away from the injury site (7/7). In the supplemented cultures, the number decreased to 6/8 in AChE and 4/8 in hemolymph, with reduced migration distances in both cases. Significance. The SDS transection approach is simple and inexpensive, yet provides flexibility in studying neuroregeneration, particularly when it is important to make sure there are no retrograde signals from the distal segments affecting regeneration. Neurons are known to not only be under tension but also balanced in terms of force, and the balance is obviously disrupted by transection. Our experimental platform, verified with Aplysia, can be extended to mammalian systems, and help us gain insight into the role that neurotrophic factors and mechanical tension play during neuronal regeneration.