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

• 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.

Keyword

axonal injury

axonal regeneration

glia cell response to injury

models of injury

oxidative stress

hydrogen peroxide

animal cell

article

astrocyte

brain cortex

cytotoxicity

embryo

glia

in vitro study

molecular mechanics

mouse

nerve cell lesion

nerve fiber growth

nerve fiber regeneration

neurite

nonhuman

traumatic brain injury

Animals

Axons

Brain Injuries

Cells

Cultured

Coculture Techniques

Disease Models

Animal

Immunohistochemistry

Mice

Microscopy

Fluorescence

Nerve Regeneration

Neuroglia

Neurons

Publication and Content Type

ref (subject category)

art (subject category)