Conditioning lesions before or after spinal cord injury recruit broad genetic mechanisms that sustain axonal regeneration: Superiority to camp-mediated effects

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Abstract

Previous studies indicate that peripheral nerve conditioning lesions significantly enhance central axonal regeneration via modulation of cAMP-mediated mechanisms. To gain insight into the nature and temporal dependence of neural mechanisms underlying conditioning lesion effects on central axonal regeneration, we compared the efficacy of peripheral sciatic nerve crush lesions to cAMP elevations (in lumbar dorsal root ganglia) on central sensory axonal regeneration when administered either before or after cervical spinal cord lesions. We found significantly greater effects of conditioning lesions compared to cAMP elevations on central axonal regeneration when combined with cellular grafts at the lesion site and viral neurotrophin delivery; further, these effects persisted whether conditioning lesions were applied prior to or shortly after spinal cord injury. Indeed, conditioning lesions recruited extensively greater sets of genetic mechanisms of possible relevance to axonal regeneration compared to cAMP administration, and sustained these changes for significantly greater time periods through the post-lesion period. We conclude that cAMP-mediated mechanisms account for only a portion of the potency of conditioning lesions on central axonal regeneration, and that recruitment of broader genetic mechanisms can extend the effect and duration of cellular events that support axonal growth.

Highlights

► Conditioning lesions are effective after spinal cord injury in combination with NT-3 gene transfer. ► Conditioning lesions have significant greater effects on axonal regeneration than increases in cAMP. ► Conditioning lesions activate more extensive transcriptional mechanisms than increases in cAMP.

Introduction

Regeneration in the injured CNS is limited by several mechanisms including the inhibitory central environment (Filbin, 2003, Schwab, 2004, Silver and Miller, 2004), a lack of growth-promoting substrates and diffusible proteins in the lesion site (Hendriks et al., 2004, Oudega and Xu, 2006), inflammatory responses (Bethea and Dietrich, 2002, Popovich and McTigue, 2009), and insufficient recruitment of intrinsic neuronal growth mechanisms (Costigan et al., 2002, Goldberg et al., 2002, Plunet et al., 2002).

One means of stimulating central axonal regeneration is the application of conditioning lesions to the peripheral branch of sensory axons, before central spinal cord lesions are placed (Neumann and Woolf, 1999, Neumann et al., 2005, Richardson and Issa, 1984). Mechanisms underlying the pre-conditioning effect on axonal regeneration have been the subject of extensive study and involve Il-6 and stat-3 signaling (Cafferty et al., 2004, Cao et al., 2006, Qiu et al., 2005), activation of transcription factors including CREB (Gao et al., 2004) and ATF3 (Seijffers et al., 2006, Seijffers et al., 2007), and cAMP-related pathways to induce protein kinase A signaling (Cai et al., 2001, Cai et al., 2002, Lu et al., 2004, Neumann et al., 2002, Nikulina et al., 2004, Qiu et al., 2002). Indeed, injection of cAMP alone has been reported to replicate the effects of peripheral conditioning lesions on central axonal regeneration (Lu et al., 2004, Neumann et al., 2002). However, it remains unclear what portion of the conditioning effect is mediated by cAMP (Gao et al., 2004, Neumann et al., 2002, Qiu et al., 2002), and whether other mechanisms are necessary or sufficient for peripheral conditioning lesions (Andersen et al., 2000, Han et al., 2004). Further, the temporal dependence of central axonal regeneration on peripheral conditioning remains incompletely understood. It has been reported that conditioning lesions or cAMP injections into dorsal root ganglia support central axonal regeneration when applied prior to, but not after, a central lesion (Neumann and Woolf, 1999, Neumann et al., 2005) but recent studies have suggested that conditioning lesions remain effective when applied up to 16 months post spinal cord injury (Kadoya et al., 2009). As peripheral pre-conditioning lesions or “post-conditioning” at stages of chronic spinal cord injury appear to recruit similar genetic mechanisms (Kadoya et al., 2009), one would, a priori, expect similar regenerative responses when conditioning lesions are applied prior to or following central lesions.

Given the importance of further understanding the specific nature and temporal dependence of the conditioning lesion effect on central axonal regeneration, we compared the relative potency of cAMP and sciatic nerve crush (conditioning lesion) on sensory axonal growth in vitro, and following cervical spinal cord injury in vivo. Further, we examined the relative potency of these approaches when conditioning lesions preceded, or followed, spinal cord lesions. The ability of cAMP increases or conditioning lesions to influence genetic mechanisms was assessed by Affymetrix whole-genome arrays and confirmed by PCR. We now report significantly greater efficacy of conditioning lesions on neuritic growth in vitro and in vivo compared to cAMP-mediated effects, retention of this efficacy whether applied before or shortly after central injury, and recruitment of extensively greater genetic mechanisms related to transcriptional activation and candidate regeneration-associated gene expression. These findings have important implications for the targeting of intraneuronal mechanisms to enhance regeneration in a time frame of practical relevance.

Section snippets

Experimental design

Effects of conditioning lesions versus cAMP were examined in explant assays of adult and postnatal dorsal root ganglion (DRG) neurons and, separately, postnatal day 7 cerebellar granule neuron cultures. In addition, we examined effects of systemic cAMP augmentation on neurite outgrowth by systemic infusions of the phosphodiesterase-IV (PDE-IV) inhibitor mesopram (Schering AG, Berlin) (Dinter et al., 2000). Neurons in both DRG and cerebellar granule cell assays were cultured either on poly-l

Statistical analysis

In all quantification procedures, multiple group comparisons were made by ANOVA with a significance criterion of p < 0.05. Post hoc differences were tested by Fisher's least square difference. Data are presented as mean ± standard error of the mean.

Conditioning lesions elicit significantly greater in vitro neurite outgrowth from adult DRG neurons than cAMP

Adult DRG neurons cultivated on poly-l-lysine and isolated from animals 3 or 7 days after conditioning lesions exhibited a significant 2-fold increase in neurite length when compared to DRG neurons from naïve animals (Figs. 1A,B). In contrast, infusion of the PDE-IV inhibitor mesopram for 3 or 7 days did not increase neurite length. Similarly, cultivation of adult DRGs in the presence of 2 mM db-cAMP did not significantly increase neurite extension on poly-l-lysine or myelin. (Fig. 1C). Thus,

Discussion

Conditioning lesions of the peripheral branch of sensory axons have long been recognized to enhance the growth capacity of central sensory neuron projections when injury of the peripheral process precedes injury in the CNS (Neumann and Woolf, 1999, Richardson and Issa, 1984). Our studies show that a single post-conditioning lesion is also effective in enhancing central axonal bridging across a spinal cord lesion site when combined with NT-3 delivery. Consistent with previous studies, NT-3

Acknowledgments

Supported by grants from the Veterans Administration, International Spinal Research Trust, the Dr. Miriam and Sheldon G. Adelson Medical Research Foundation, the Bernard and Anne Spitzer Charitable Trust, Craig H. Neilson Foundation and the NIH (NS054833, NS049881, and NS047101). We thank Fuying Gao for assistance with data analysis.

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