In vitro models of axon regeneration

Abstract

A variety of in vitro models have been developed to understand the mechanisms underlying the regenerative failure of central nervous system (CNS) axons, and to guide pre-clinical development of regeneration-promoting therapeutics. These range from single-cell based assays that typically focus on molecular mechanisms to organotypic assays that aim to recapitulate in vivo behavior. By utilizing a combination of models, researchers can balance the speed, convenience, and mechanistic resolution of simpler models with the biological relevance of more complex models. This review will discuss a number of models that have been used to build our understanding of the molecular mechanisms of CNS axon regeneration.

Keywords: Axon regeneration; Cell based assays; High content screening; In vitro models; Neurite outgrowth; Organotypic assays.

Figure 1. Examples of cell-based in vitro models of axon regeneration from neurite outgrowth experiments

Micrographs show various types of primary neurons cultured in the absence (A-A’, C-C’, E-E’, G-G’) or presence (B-B’, D-D’, F-F’, H-H’) of a neurite growth promoting kinase inhibitor, RO1A03. A-H) Show anti-β3 Tubulin immunostaining used to visualize the neuronal cytoskeleton (green). A’-H’) Automated tracing of neuronal cytoskeleton (grey) obtained using the Cellomics Neuronal Profiler bioapplication shows cell bodies of valid neurons (blue), neurites (orange and purple), and cells rejected by the tracing algorithm (red) based on morphological parameters. A-B) Postnatal (P3) rat cortical neurons 2 DIV. C-D) Postnatal (P21) retinal ganglion cells 3 DIV. E-F) Embryonic (E18) rat hippocampal neurons 2DIV. G-H) Adult (10w) mouse dorsal root ganglion neurons 1 DIV. Images are single fields obtained with Cellomics VTI (5× objective) from cells cultured in 96-well plates. Scale bar is 200μm.