Electrical stimulation of intact peripheral sensory axons in rats promotes outgrowth of their central projections

https://doi.org/10.1016/j.expneurol.2007.11.007Get rights and content

Abstract

A lesion of a peripheral nerve before a second injury (conditioning lesion, CL), enhances peripheral and central regeneration of dorsal root ganglion (DRG) axons. This effect is mediated by elevated neuronal cAMP. Here we wanted to investigate whether electrical stimulation (ES) of an intact nerve, which has been shown to accelerate peripheral axon outgrowth, is also effective in promoting axon regeneration of injured DRG axons in vitro and of the central DRG axons in vivo and, whether this effect is mediated by elevation of cAMP. For the in vitro assay, the intact sciatic nerve of adult rats was stimulated at 20 Hz for 1 h, 7 days before harvest and primary culture of DRG neurons on a growth permissive substrate. In the in vivo study, the central axons of the lumbosacral DRGs were cut in the Th8 dorsal column, and the sciatic nerve was either cut or left intact, and subjected to 1 h ES at 20 Hz or 200 Hz. In vitro, ES increased neurite outgrowth 4-fold as compared to non-stimulated DRG neurons. In vivo, ES at 20 Hz significantly increased axon outgrowth into the central lesion site as compared to the Sham control. The 20 Hz ES was as effective as the CL in increasing axon outgrowth into the lesion site but not in promoting axonal elongation even though 20 Hz ES increased intracellular cAMP levels in DRG neurons as effectively as the CL. Thus elevation of cAMP may account for the central axonal outgrowth after ES and a CL.

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.

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