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

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 12 hours 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.

Cytoskeletal disruption activates the DLK/JNK pathway, which promotes axonal regeneration and mimics a preconditioning injury

“…We wished to know whether cytoskeletal disruption activates the DLK/JNK pathway in adult neurons, and whether this activation is sufficient to promote axon regeneration. The dissection and plating of the neurons is an injury (Frey et al, 2015; Saijilafu et al, 2013; Zou et al, 2009), and we find that after one day in culture pcJun levels are high (Figure 6A–C). This is consistent with in vivo results demonstrating that a crush injury induces the phosphorylation of cJun in the DRGs (Shin et al, 2012).…”

“…We have established an in vitro paradigm in which adult DRG neurons have recovered from the injury of being dissected from the animal (this new assay is characterized in detail in Frey et al, 2015). These neurons have low levels of the injury marker pcJun, and upon replating have limited capacity to regrow axons.…”

The nerve conditioning lesion: A strategy to enhance nerve regeneration

“…Although initial methodologies precluded clinical translatability, research over the past 4 decades has yielded valuable mechanistic insights. Such insights have not only provided a solid foundation to support the use of CLs in clinical settings, but they have helped to identify molecular targets that could potentially be pharmacologically manipulated to enhance peripheral nerve regeneration …”

Primary Postnatal Dorsal Root Ganglion Culture from Conventionally Slaughtered Calves

“…Loss of pseudounipolarity has been described also in cultured DRG neurons from rat, mice and rabbit and have been attributed to different factors including the removal of satellite cells, which prevent dendritic formation in vivo [8], the type of GF supplementation [57] and activation of transcription factors associated to the regenerative state of dissociated neurons [58]. As in DRG cultures from other species [47, 59] postnatal bovine DRG neurons still had significant numbers of ensheathing cells attached, which does not support the removal of satellite cells as the cause for multipolarity.…”