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

• Published in: Experimental neurology 2015-01, Vol.263, p.350-363
• Main Authors: Frey, E., Valakh, V., Karney-Grobe, S., Shi, Y., Milbrandt, J., DiAntonio, A.
• Format: Article
• Language: English
• Subjects: Animals; Axon regeneration; Cells, Cultured; cJun N-terminal kinase (JNK); Dorsal root ganglion (DRG); Dual leucine zipper kinase (DLK); Ganglia, Spinal - physiology; Immunohistochemistry; In Vitro Techniques; Mice; Mice, Inbred C57BL; Nerve Regeneration - physiology; Real-Time Polymerase Chain Reaction; Replating assay; Sensory Receptor Cells - physiology; Superior cervical ganglion 10 (SCG10)
• ISSN: 0014-4886; 1090-2430
• DOI: 10.1016/j.expneurol.2014.10.012

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. •Dissection and dissociation of DRG neurons activates a pro-regenerative response.•The regenerative response fades in culture resulting in functionally naïve neurons.•Injury or Forskolin re-induce the regenerative response of functionally naïve neurons.•Assay can identify drugs that induce regenerative response in the absence of injury.