An in vitro assay to study induction of the regenerative state in sensory neurons

Exp Neurol. 2015 Jan;263:350-63. doi: 10.1016/j.expneurol.2014.10.012. Epub 2014 Nov 4.

Abstract

After injury, peripheral neurons activate a pro-regenerative program that facilitates axon regeneration. While many regeneration-associated genes have been identified, the mechanism by which injury activates this program is less well understood. Furthermore, identifying pharmacological methods to induce a pro-regenerative state could lead to novel treatments to repair the injured nervous system. Therefore, we have developed an in vitro assay to study induction of the pro-regenerative state following injury or pharmacological treatment. First, we took advantage of the observation that dissociating and culturing sensory neurons from dorsal root ganglia activates a pro-regenerative program. We show that cultured neurons activate transcription factors and upregulate regeneration-associated genes common to the pro-regenerative program within the first hours after dissection. In a paradigm similar to pre-conditioning, neurons injured by dissociation display enhanced neurite outgrowth when replated as early as 12h after being removed from the animal. Furthermore, stimulation of the pro-regenerative state improves growth on inhibitory substrates and requires DLK/JNK signaling, both hallmarks of the pro-regeneration response in vivo. Finally, we modified this assay in order to identify new methods to activate the pro-regenerative state in an effort to mimic the pre-conditioning effect. We report that after several days in culture, neurons down-regulate many molecular hallmarks of injury and no longer display enhanced neurite outgrowth after replating. Hence, these neurons are functionally naïve and are a useful tool for identifying methods to induce the pro-regenerative state. We show that both injury and pre-treatment with forskolin reactivate the pro-regenerative state in this paradigm. Hence, this assay is useful for identifying pharmacological agents that induce the pro-regenerative state in the absence of injury.

Keywords: Axon regeneration; Dorsal root ganglion (DRG); Dual leucine zipper kinase (DLK); Replating assay; Superior cervical ganglion 10 (SCG10); cJun N-terminal kinase (JNK).

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cells, Cultured
  • Ganglia, Spinal / physiology*
  • Immunohistochemistry
  • In Vitro Techniques
  • Mice
  • Mice, Inbred C57BL
  • Nerve Regeneration / physiology*
  • Real-Time Polymerase Chain Reaction
  • Sensory Receptor Cells / physiology*