Increased chondroitin sulfate proteoglycan expression in denervated brainstem targets following spinal cord injury creates a barrier to axonal regeneration overcome by chondroitinase ABC and neurotrophin-3
Introduction
For regeneration of injured mammalian CNS axons to be meaningful, functional synapses must be restored with target neurons. Increasing evidence suggests that following CNS lesions, several chemical mediators of gliosis that accompany the inflammatory response following breakdown of the blood brain barrier (Johns et al., 1992, Fitch and Silver, 1997, Rhodes and Fawcett, 2004, Silver and Miller, 2004) locally stimulate the increased expression of a number of chondroitin sulfate proteoglycans (CSPGs) (McKeon et al., 1995, Asher et al., 2000, Smith and Strunz, 2005). The increased expression of CSPGs in and around brain or spinal cord injury (SCI) sites is a major contributor to the failure of spontaneous axonal regeneration (McKeon et al., 1991, Fitch and Silver, 1997, Bruce et al., 2000, Jones et al., 2002, Jones et al., 2003, Tang et al., 2003). The mechanism by which these macromolecules inhibit regenerating axons is incompletely understood but may be the result of disrupted adhesive interactions (Grumet et al., 1996, Milev et al., 1998), decreased diffusion of secreted molecules (Gruskin et al., 2003) and altered intracellular signaling (Sivasankaran et al., 2004, Zhou et al., 2006). In the absence of CSPGs (Bradbury et al., 2002, Davies et al., 2004, Grimpe and Silver, 2004) or following the application of neurotrophins (Oudega and Hagg, 1996, Bradbury et al., 1999, Oudega and Hagg, 1999, Lu et al., 2004, Lu et al., 2007, Taylor et al., 2006), injured sensory axons are able to regenerate past SCI sites and through degenerating myelin debris (Bradbury et al., 1999, Bradbury et al., 2002, Davies et al., 1999, Davies et al., 2004).
Following CNS injury, inflammatory activation of astrocytes and microglia is not solely limited to areas immediately surrounding the sites of trauma but is also present around denervated target neurons (Koshinaga and Whittemore, 1995, Deller and Frotscher, 1997, Aldskogius and Kozlova, 1998, Liu et al., 1998). It is not known whether these activated glial cells intermingled between neurons denervated by SCI (Koshinaga and Whittemore, 1995) might be capable of actually preventing regenerating sensory axons in the dorsal columns from re-entering their proper synaptic targets by further saturating the extracellular matrix with newly secreted CSPGs. In the normal adult CNS, CSPGs can contribute to the formation of the specialized lattice-like perineuronal net that cloaks neuronal cell bodies and is thought to inhibit synaptic plasticity (Golgi, 1893, Svensson et al., 1984, Lander et al., 1997, Bruckner et al., 1998, Takahashi-Iwanaga et al., 1998, Pizzorusso et al., 2002, Corvetti and Rossi, 2005). We recently demonstrated that collateral sprouting by spared primary afferents inside the partially denervated adult rat brainstem cuneate nucleus following cervical SCI is inhibited by CSPGs within the nucleus and is promoted by their local digestion (Massey et al., 2006). Here, using a combination of quantitative real-time polymerase chain reaction, immunoblotting and immunohistochemistry we show, for the first time, that cervical SCI results in the significantly increased expression of the CSPGs NG2, neurocan, and brevican in the distant denervated dorsal column nuclei (DCN). Furthermore, as is the case at sites of direct CNS injury, these CSPGs present a potent barrier to reinnervation by regenerating axons from microtransplanted adult sensory neurons following SCI that can be overcome by chondroitinase ABC (chABC) application and increased neurotrophin-3 (NT-3) expression within the nucleus. Portions of these results have been previously reported in abstract form (Massey et al., 2005).
Section snippets
Materials and methods
All animal manipulation procedures complied with NIH regulations and were approved by the Institutional Animal Care and Use Committee guidelines at both the University of Louisville and Case Western Reserve University School of Medicine. Adult male (275–350 g) Sprague-Dawley rats were used for these experiments. Each rat was anesthetized with an intramuscular injection of a mixture of ketamine hydrochloride (50 mg/kg), xylazine (6.5 mg/kg) and acepromazine (2.5 mg/kg) for each surgical
Results
Successful treatments developed to repair axonal pathways damaged by SCI ultimately will need to result in regeneration and functional reinnervation of target neurons. Our goal was to investigate whether the gliosis induced by denervation of the adult rat DCN following SCI is accompanied by increased CSPG expression that inhibits anatomical axonal regeneration.
Discussion
New synaptic contacts must be made for functional improvement after SCI. CSPGs limit both CNS axonal regeneration and functional plasticity (Davies et al., 1997, Davies et al., 1999, McKeon et al., 1991, Fitch and Silver, 1997, Bradbury et al., 2002, Grimpe and Silver, 2004, Pizzorusso et al., 2002, Pizzorusso et al., 2006, Barritt et al., 2006, Houle et al., 2006, Massey et al., 2006). Here, we investigated whether denervation of brainstem spinal cord targets induces an increase of CSPG
Conclusion
Because of the considerable difficulty that has been encountered attempting to develop treatment strategies that promote successful axonal regeneration of severed axons back to target neurons denervated by SCI, much of the work has justifiably focused on circumventing the barriers erected at the injury site. Our findings demonstrate that additional challenges await approaching axons once they arrive at their desired targets. These findings further suggest that proper reconstruction and
Acknowledgments
This work was supported by the National Institutes of Health NS40411 (S.M.O.), RR15576 (S.M.O.), NS25713 (J.S.), and RR16481 (N.G.F.C.). J.M.M. was supported by a fellowship awarded from the Kentucky National Science Foundation/Experimental Program to Stimulate Competitive Research EPS-9874764 (N.G.F.C.) and by the Summer Research Scholars Program at the School of Medicine in the University of Louisville. The authors extend their appreciation to the Stan Gerson (Case Western Reserve University)
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The laboratories of these investigators contributed equally to this project.