In vitro priming response in dorsal root ganglia partially mimics injury-driven pre-conditioning response and reprograms neurons for enhanced outgrowth
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.
Section snippets
Animals and axotomy-induced pre-conditioning
Adult male SD rats of 4–6 weeks were used for the study. Axotomy-induced pre-conditioning (in vivo priming) of DRGs was done as described previously (Krishnan et al., 2018a). Briefly, the right sciatic nerve was completely transected under aseptic conditions. The animals were allowed to survive for three days and maintained under buprenorphine (0.05 mg/kg) for analgesia. After three days, the animals were euthanized using high dose isoflurane and the lumbar DRGs (L4-L6) from the ipsilateral
In vitro priming promotes the intrinsic growth potential of adult sensory neurons
In order to examine if an overnight incubation of normal DRGs in vitro simulates an axotomy-driven pre-conditioning response, we incubated whole lumbar DRGs isolated from healthy rats for 24 h–72 h at 37o C in an incubator maintained at 5% CO2. We used two compositions of growth media for the DRG incubation; a) containing 10% FBS, b) conventional media containing the growth factors N2 supplement and NGF, herein referred as FBS and N2/NGF media respectively. After incubation, the individual
Discussion
Although supplementation of neurotrophic factors improves axon regrowth in animal models of nerve injuries, this approach has not been successful in treating neurodegenerative disorders in clinical settings (Weissmiller and Wu, 2012). Hence, combining neurotrophic factors with additional growth promoters may be worthwhile and promising for nerve repair. Studies in the past have identified many potential molecular targets for nerve regeneration, most of which are at the pre-clinical stage and it
Conclusion
Overall, our novel model partially mimics a pre-conditioning response in DRGs, especially at the molecular and growth response level. However, the model tells its own story too, by demonstrating an altered activation pattern of SGCs and phenotypic distribution changes of sensory neurons. Nevertheless, this model represents molecular and cellular changes that facilitate neurite outgrowth. Systematic understanding of the molecular and cellular changes associated with this new approach would
CRediT authorship contribution statement
Anand Krishnan: Conceptualization, data collection, analysis, interpretation of data, and manuscript draft preparation. Shubham Dwivedi: Data collection, analysis. Ambika Chandrasekhar: Data collection, analysis. Aparna Areti: Data collection and analysis. Douglas Zochdone: Conceptualization, guidance of the study, interpretation of data, revision of the manuscript draft and final preparation of manuscript.
Funding
This work was supported by the Canadian Institutes of Health Research (CIHR) funding of the Zochodne laboratory [Grant No. FRN148675]. This work was also supported by the research start-up fund from the College of Medicine, University of Saskatchewan, to the Krishnan laboratory.
References (31)
c-Jun reprograms Schwann cells of injured nerves to generate a repair cell essential for regeneration
Neuron
(2012)Intraganglionic interactions between satellite cells and adult sensory neurons
Mol. Cell. Neurosci.
(2015)Endogenous BDNF regulates induction of intrinsic neuronal growth programs in injured sensory neurons
Exp. Neurol.
(2010)Nerve regeneration: understanding biology and its influence on return of function after nerve transfers
Hand Clin.
(2016)Intrinsic facilitation of adult peripheral nerve regeneration by the Sonic hedgehog morphogen
Exp. Neurol.
(2015)- et al.
Regeneration of dorsal column fibers into and beyond the lesion site following adult spinal cord injury
Neuron
(1999) - et al.
Signaling over distances
Mol. Cell. Proteomics
(2016) Peripheral neuron plasticity is enhanced by brief electrical stimulation and overrides attenuated regrowth in experimental diabetes
Neurobiol. Dis.
(2015)Activating transcription factor 3 (ATF3) induction by axotomy in sensory and motoneurons: a novel neuronal marker of nerve injury
Mol. Cell. Neurosci.
(2000)Rapamycin-resistant mTOR activity is required for sensory axon regeneration induced by a conditioning lesion
eNeuro
(2016)
PTEN inhibition to facilitate intrinsic regenerative outgrowth of adult peripheral axons
J. Neurosci.
Enhancing adult nerve regeneration through the knockdown of retinoblastoma protein
Nat. Commun.
Expression and manipulation of the APC-beta-catenin pathway during peripheral neuron regeneration
Sci. Rep.
Beyond trophic factors: exploiting the intrinsic regenerative properties of adult neurons
Front. Cell. Neurosci.
Differential expression of mRNAs for neurotrophins and their receptors after axotomy of the sciatic nerve
J. Cell Biol.
Cited by (4)
Self-renewing macrophages in dorsal root ganglia contribute to promote nerve regeneration
2023, Proceedings of the National Academy of Sciences of the United States of AmericaSurvival of compromised adult sensory neurons involves macrovesicular formation
2022, Cell Death Discovery