In vitro priming response in dorsal root ganglia partially mimics injury-driven pre-conditioning response and reprograms neurons for enhanced outgrowth

Highlights

• Peripheral nerve injury pre-conditions sensory neurons for regeneration.

• We generated an in vitro model that partially mimics the pre-conditioning response.

• This model shows non-classical molecular changes supporting neuron regeneration.

• This in vitro model will enable analysis of the molecular basis of neuron regeneration.

Abstract

Peripheral nerve injuries have the potential to bring about long-term disabilities in individuals. The major issue in repairing nerve injuries is the poor growth rate of axons. Although several molecules have been identified as potential candidates for improving axon growth, their potential translation into clinical practice is preliminary and largely unexplored. This necessitates identifying additional molecular candidates with superior potential to improve axon growth. Lack of a simple non-surgical screening model also poses a hurdle in rapidly screening potential candidate molecules. In this work, we developed a novel, rapid screening model for nerve regeneration therapeutics that retains a focus on adult neurons. The model involves simple incubation of sensory ganglia over a period of 24 h prior to dissociation. Surprisingly, this model features unique events that reprogram both sensory neurons and supporting glia favoring axon growth. Moreover, several associated cellular and molecular changes involved in this model partially mimic classic axotomy-induced changes in sensory ganglia. Overall, this model presents with a platform that not only allows rapid screening of drug candidates but offers opportunities in studying novel intrinsic molecular changes in both neurons and glial cells directed towards improving the pace of axon growth.

Introduction

Peripheral nerve injuries can occur due to accidents, sports injuries, or medical conditions, and lead to loss of essential functions. Surgical intervention is the only clinical treatment of choice at present (Hussain et al., 2020). However, although surgery can reconnect injured nerves, the poor growth rate of axons maintains denervation of target muscles for an extended period resulting in irreversible muscular atrophy and permanent disability (Gordon, 2016; Zochodne, 2012). Therefore, novel strategies that can improve the pace of axon growth should be identified to manage peripheral nerve injuries.

Previous research had identified a family of proteins called neurotrophic factors with the ability to improve axon growth. This family of proteins includes NGF, BDNF, NT3, NT4/5, CNTF, and others (Duraikannu et al., 2019; Verge et al., 1996). Axotomy induces the expression of neurotrophic factors in sensory ganglia. The collective events of axotomy-induced changes in neurotrophic factors and other growth regulatory molecules in the sensory ganglia, which prime the neurons for regeneration, is called ‘pre-conditioning’, or ‘in vivo priming’ of neurons (Neumann and Woolf, 1999). Classically, neurons and supporting satellite glial cells (SGCs) both undergo priming after axotomy (Christie et al., 2015). The in vivo priming model has been extensively used to understand the trophic actions of neurotrophic factors and other growth regulatory molecules (Krishnan et al., 2018a; Martinez et al., 2015; Geremia et al., 2010). This model has also enabled the identification of growth suppressors and neurodegenerative molecules in regenerating neurons (Christie et al., 2010; Christie et al., 2014; Duraikannu et al., 2018).

The in vivo priming model requires animals to undergo axotomy and survival for at least three days for the priming to occur. Here, we examined the utility of a novel model to replace the axotomy-induced priming model and to avoid the morbidity of a pre-harvesting axotomy. In our new model, the DRGs were primed in vitro by simple incubation without a prior axotomy procedure. Interestingly, we found that in vitro priming accelerates the growth response in neurons, partially mimicking in vivo pre-conditioning response. The approach appears to reprogram both neurons and SGCs in a non-classic manner, indicating that this model also has the potential to reveal additional novel molecular targets for nerve regeneration.