Electrical stimulation of intact peripheral sensory axons in rats promotes outgrowth of their central projections
Introduction
Injured axons in the central nervous system (CNS) fail to regenerate (Schwab and Bartholdi, 1996, Silver and Miller, 2004) in contrast to those in the peripheral nervous system (PNS) (Fu and Gordon, 1997). This disparity is illustrated by the capacity of injured peripheral but not central axons of dorsal root ganglion (DRG) sensory neurons to regenerate. Axonal injury in the PNS but not the CNS, leads to pronounced expression of growth associated genes (Broude et al., 1997, Schreyer and Skene, 1993). Moreover, the injured CNS has an abundance of inhibitory molecules such as myelin associated proteins (Filbin, 2003) and proteoglycans at the glial scar (Busch and Silver, 2007) that are an impediment to axonal regeneration.
Experimental strategies to promote axonal regeneration in the CNS include attempts to create a growth permissive environment (Fouad and Pearson, 2004, Houle et al., 2006), and/or altering the neuronal response to inhibitory molecules (Domeniconi and Filbin, 2005). For example, the neuronal regenerative response has been altered by elevating intra-neuronal cAMP levels pharmacologically or by a conditioning lesion (CL) of DRG sensory neurons (Neumann et al., 2002, Qiu et al., 2002, Cai et al., 1999, Dergham et al., 2002, Lehmann et al., 1999). The CL, a lesion of a peripheral axons before a second injury, enhances peripheral (McQuarrie and Grafstein, 1973, McQuarrie et al., 1977) and central (Neumann and Woolf, 1999, Richardson and Issa, 1984) axonal regeneration. Recently, a relatively noninvasive 1 h period of electrical stimulation (ES) proximal to a site of transection and surgical repair of a pheripheral nerve has also been shown to accelerate axonal outgrowth across the injury site (Al-Majed et al., 2000b, Brushart et al., 2002). This effect has been related to an up-regulation of regeneration associated genes (Al-Majed et al., 2000a, Al-Majed et al., 2004). In this study, we asked whether ES of the intact sciatic nerve also promotes regeneration of DRG axons in the CNS after a dorsal column lesion. First, we determined the effects of ES on neurite growth in an in vitro preparation of DRG neurons, and second, we examined and compared the effects of sciatic ES and a CL on the regeneration of central axons of DRG sensory neurons in an in vivo model of spinal cord injury. In order to test a clinically relevant scenario, we applied ES immediately following the central lesion of DRG axons rather than a week before. This was despite the fact that a CL of the DRG neurons at the same time as the central lesion is not as effective as a CL 7 days before (Neumann and Woolf, 1999). We found that ES enhanced neurite and axonal outgrowth in vitro and in vivo and that this effect was associated with increased intracellular cAMP levels in the DRG sensory neurons. ES was not as effective as a CL in promoting elongation of the regenerating axons further into the lesion site but the advantage of ES over a CL is its clinical feasibility because ES can be applied to an intact nerve and conditions the neuron without cutting its axon.
Section snippets
Materials and methods
Experiments were performed on adult female Sprague Dawley rats (200–220 g), and approved by local authorities according to the Canadian Council for Animal Care guidelines. All surgeries were performed under Ketamine (Vetalar, Bioniche, Belleville, Ontario) and Xylacine (Rompun, Bayer, Toronto, Ontario) anesthesia.
ES promotes neurite outgrowth in vitro
The capacity of dissociated adult rat DRG sensory neurons to grow neurites when plated on a permissive substrate (Lindsay, 1988) can be accelerated if a CL has been applied previously (Smith and Skene, 1997). Here we wanted to determine if ES of the intact sciatic nerve can mimic this CL effect.
One or 7d after ES of the right sciatic nerve, DRGs were harvested and the sensory neurons whose axons projected into the left non-stimulated and right stimulated sciatic nerves were plated for 24 h on a
Discussion
In this study we demonstrated that a single ES session of an intact peripheral nerve for 1 h enhances neurite outgrowth of DRG neurons in vitro, outgrowth of the central sensory axons into the lesion site in vivo, and elevates intracellular cAMP levels in the DRG neurons. ES has been shown to promote axonal outgrowth in the PNS (Al-Majed et al., 2000b, Brushart et al., 2002, Nix and Hopf, 1983) but, in contrast to the CL, did not accelerate the rate of axonal regeneration (Bisby and Pollock,
Acknowledgments
We would like to thank N. Tyreman and R. Vavrek for their helpful technical support and Dr X. Navarro for his helpful editorial suggestions. The work was supported by an operating grant to TG from the Canadian Institutes of Health Research. EU was a recipient of a research fellowship from the Spanish Ministry of Education and Sciences. JS is funded by the NINDS/NS25713.
References (53)
- et al.
c-Jun expression in adult rat dorsal root ganglion neurons: differential response after central or peripheral axotomy
Exp. Neurol.
(1997) - et al.
The role of extracellular matrix in CNS regeneration
Curr. Opin. Neurobiol.
(2007) - et al.
Prior exposure to neurotrophins blocks inhibition of axonal regeneration by MAG and myelin via a cAMP-dependent mechanism
Neuron
(1999) - et al.
Overcoming inhibitors in myelin to promote axonal regeneration
J. Neurol. Sci.
(2005) - et al.
Restoring walking after spinal cord injury
Prog. Neurobiol.
(2004) - et al.
Electrical stimulation promotes sensory neuron regeneration and growth-associated gene expression
Exp. Neurol.
(2007) - et al.
Cyclic AMP elevates tubulin expression without increasing intrinsic axon growth capacity
Exp. Neurol.
(2004) - et al.
Chronic infusion of agents that increase cyclic AMP concentration enhances the regeneration of mammalian peripheral nerves in vivo
Exp. Neurol.
(1987) - et al.
Effect of a conditioning lesion on optic nerve regeneration in goldfish
Brain Res.
(1981) - et al.
Effect of acetoxycycloheximide and dibutyryladenosine cyclic 3′:5′-monophosphate on axonal regeneration in the goldfish optic nerve
Brain Res.
(1983)
Axonal regeneration in the rat sciatic nerve: effect of a conditioning lesion and of dbcAMP
Brain Res.
Regeneration of dorsal column fibers into and beyond the lesion site following adult spinal cord injury
Neuron
Regeneration of sensory axons within the injured spinal cord induced by intraganglionic cAMP elevation
Neuron
Electrical stimulation of regenerating nerve and its effect on motor recovery
Brain Res.
Spinal axon regeneration induced by elevation of cyclic AMP
Neuron
Evidence against cholera toxin B subunit as a reliable tracer for sprouting of primary afferents following peripheral nerve injury
Brain Res.
Actions of neuropoietic cytokines and cyclic AMP in regenerative conditioning of rat primary sensory neurons
Exp. Neurol.
Electrical stimulation accelerates and increases expression of BDNF and trkB mRNA in regenerating rat femoral motoneurons
Eur. J. Neurosci.
Brief electrical stimulation promotes the speed and accuracy of motor axonal regeneration
J. Neurosci.
Electrical stimulation accelerates and enhances expression of regeneration-associated genes in regenerating rat femoral motoneurons
Cell. Mol. Neurobiol.
Peripheral axotomy induces only very limited sprouting of coarse myelinated afferents into inner lamina II of rat spinal cord
Eur. J. Neurosci.
Increased regeneration rate in peripheral nerve axons following double lesions: enhancement of the conditioning lesion phenomenon
J. Neurobiol.
NT-3 promotes growth of lesioned adult rat sensory axons ascending in the dorsal columns of the spinal cord
Eur. J. Neurosci.
Electrical stimulation of the corticospinal tract promotes growth of spared axons after partial injury
Electrical stimulation promotes motoneuron regeneration without increasing its speed or conditioning the neuron
J. Neurosci.
Rho signaling pathway targeted to promote spinal cord repair
J. Neurosci.
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2020, Experimental NeurologyCitation Excerpt :One promising strategy is electrical stimulation (ES) of peripheral nerves at the time of nerve repair, which has been shown to improve motor and sensory nerve regeneration in animal models of acute and chronic peripheral nerve injury (Al-Majed et al., 2000a; Elzinga et al., 2015; Geremia et al., 2007; Gordon et al., 2009a; Gordon and English, 2016; Shapira et al., 2019; Willand et al., 2016; Xu et al., 2014). The retrograde conduction of action potentials from the site of ES to the neuronal cell body in the spinal cord or dorsal root ganglia (DRG) increases intraneuronal cyclic AMP and induces a more rapid and sustained upregulation of regeneration associated genes (Al-Majed et al., 2000a; Al-Majed et al., 2000b; Al-Majed et al., 2004; Udina et al., 2008). These, in turn, lead to the acceleration of axonal outgrowth across an injury-repair site (Brushart et al., 2002; Udina et al., 2008), remyelination of regenerating axons (Hu et al., 2019; Wan et al., 2010), and reinnervation of end organs, even after delayed surgical repair (Al-Majed et al., 2004; Eberhardt et al., 2006; Elzinga et al., 2015; Geremia et al., 2007; Vivo et al., 2008; Xu et al., 2014).