A novel in vitro injury model based on microcontact printing demonstrates negative effects of hydrogen peroxide on axonal regeneration both in absence and presence of glia.

Abstract The molecular processes involved in axonal regeneration after traumatic brain injury (TBI) are still not fully understood. In this study, we have established a novel in vitro injury model of TBI based on microcontact printing (µCP) that enables close-up investigations of injured neurons. The model is also suitable for quantitative measurements of axonal outgrowth, making it a useful tool in the studies of basic mechanisms behind axonal regeneration. Cortical neurons from mouse embryos are cultured on µCP cover-slips for 8 days, and the neurons are then injured in a precise manner using a thin plastic tip that does not affect the µCP pattern of extracellular matrix proteins. By close-up time-lapse experiments and immunostainings, we show that the neurons have a tremendous capacity to regenerate their neurites after injury. The cut induces growth cone formation, and the regenerating axons strictly follow the µCP pattern. Moreover, by using the injury model, we demonstrate that hydrogen peroxide (H2O2) decreases axonal regeneration after injury without affecting the neurons' ability to form growth cones. Co-culture with glial cells does not rescue the axonal regeneration, indicating that the mechanism by which H2O2 affects axonal regeneration differ from its cytotoxic effect.