Rolipram-induced elevation of cAMP or chondroitinase ABC breakdown of inhibitory proteoglycans in the extracellular matrix promotes peripheral nerve regeneration

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Abstract

The inhibitory growth environment of myelin and extracellular matrix proteoglycans in the central nervous system may be overcome by elevating neuronal cAMP or degrading inhibitory proteoglycans with chondroitinase ABC (ChABC). In this study, we asked whether similar mechanisms operate in peripheral nerve regeneration where effective Wallerian degeneration removes myelin and extracellular proteoglycans slowly. We repaired transected common peroneal (CP) nerve in rats and either elevated cAMP in the axotomized neurons by subcutaneous rolipram, a specific inhibitor of phosphodiesterase IV, and/or promoted degradation of proteoglycans in the distal nerve stump by local ChABC administration. Rolipram treatment significantly increased the number of motoneurons that regenerated axons across the repair site at 1 and 2 weeks, and increased the number of sensory neurons that regenerated axons across the repair site at 2 weeks. Local application of ChABC had a similar effect to rolipram treatment in promoting motor axon regeneration, the effect being no greater when rolipram and ChABC were administered simultaneously. We conclude that blocking inhibitors of axon regeneration by elevating cAMP or degrading proteoglycans in the distal nerve stump promotes peripheral axon regeneration after surgical repair of a transected nerve. It is likely that elevated cAMP is sufficient to encourage axon outgrowth despite the inhibitory growth environment such that simultaneous enzymatic proteoglycan degradation does not promote more axon regeneration than either elevated cAMP or proteoglycan degradation alone.

Introduction

Axons in the peripheral (PNS) but not the central nervous system (CNS) regenerate after injury (Fu and Gordon, 1995a,b, 1997). Nevertheless there is only a short window of opportunity for effective PNS regeneration to occur, whereby regenerative success of injured nerves progressively decreases after prolonged axotomy and Schwann cell denervation (Fu and Gordon, 1995aFu and Gordon, 1995b, Gordon et al., 2003, Sulaiman and Gordon, 2000). Axon outgrowth from the proximal stump of transected and surgically repaired nerves is a slow process (Brushart et al., 2002) and this lengthy period when regenerating axons wander in the suture site of surgically repaired peripheral nerves (Cajal, 1928, Witzel et al., 2005) accounts, at least in part, for the delays of several weeks for all regenerating axons to cross a repair site (Brushart et al., 2002). Chondroitin sulfate proteoglycans (CSPGs) of the extracellular matrix and myelin-associated inhibitors, both potent inhibitors of neuronal regeneration in the CNS (Mueller, 1999, Sandvig et al., 2004, Tang, 2003), are also present in the peripheral nerve. These molecules are up-regulated after nerve injury, show neurite-inhibitory activity (Braunewell et al., 1995, Shen et al., 1998, Zuo et al., 1998), and may play a role in delayed axon outgrowth following peripheral nerve injury. The prolonged time course of weeks for effective removal of myelin debris by macrophages and Schwann cells (Avellino et al., 1995, Fansa and Keilhoff, 2003, George and Griffin, 1994, Stoll et al., 1989) and for degradation of glycoproteins of the extracellular matrix (Hughes et al., 2002) may account for the staggered outgrowth of axons from the proximal stump of an injured nerve (Al-Majed et al., 2000, Gordon et al., 2003).

Injured nerves in the CNS may be stimulated to regenerate their axons despite the inhibitory environment by interfering with signaling pathways that are activated by inhibitory myelin associated molecules (Cai et al., 1999, Dergham et al., 2002, Lehmann et al., 1999, Neumann and Woolf, 1999, Neumann et al., 2002, Qiu et al., 2002). The inhibition can be overcome by increasing neuronal cAMP levels in vivo (Cai et al., 1999, Dergham et al., 2002, Lehmann et al., 1999, Neumann and Woolf, 1999, Neumann et al., 2002, Qiu et al., 2002) and in vitro (Cai et al., 1999) and by selectively cleaving glycosaminoglycan (GAG) side chains from the protein core of proteoglycans with chondroitinase ABC (ChABC) (Fawcett and Asher, 1999). However, applying these pharmacological approaches to PNS regeneration has generated conflicting data. Some have reported that cAMP does promote axon outgrowth (Gershenbaum and Roisen, 1980, Kilmer and Carlsen, 1987, Pichichero et al., 1973) while others have failed to demonstrate increased axon outgrowth (Black and Lasek, 1979, Han et al., 2004, McQuarrie et al., 1977). The use of ChABC in the PNS has been shown to accelerate axon outgrowth into distal nerve stumps and acellular nerve grafts in rats (Krekoski et al., 2001, Zuo et al., 2002). Analyses of the distance of regenerating axons of thy-1 TFP-H transgenic mice demonstrated longer axon profiles in nerve grafts from wild-type littermates with ChABC, which specifically degrades CSPGs but not other proteoglycans such as heparin sulfate proteoglycans (Groves et al., 2005).

Using retrograde labeling techniques, we aim to further investigate whether elevation of cAMP or removal of GAG side chains from inhibitory proteoglycans in axotomized motor and sensory neurons in the peripheral nerve promotes PNS regeneration. We also question whether a combinational strategy of rolipram and ChABC that has proven to be effective in CNS regeneration (Fouad et al., 2005, Houle et al., 2006, Tropea et al., 2003) is also effective in the PNS.

Section snippets

Material and methods

All experiments were performed on adult female Sprague–Dawley rats (200–220 g) and approved by local authorities (Health Sciences Laboratory Animal Services, University of Alberta) according to the Canadian Council for Animal Care guidelines.

Rolipram delivery accelerates motor and sensory nerve regeneration

In order to increase cellular levels of cAMP pharmacologically in rat motor and sensory neurons, we infused rolipram systemically at a rate of 0.4 μmol/kg/h (Nikulina et al., 2004) to inhibit PDE IV, the most common isoform of phosphodiesterase in neural tissue (Jin et al., 1999). One week after transecting and repairing the nerve, a thin regenerative cord connecting the proximal and the distal stumps was observed through the silicone guide. After 2–3 weeks, there was more connective tissue

Discussion

Local inhibitors of regeneration in the distal nerve stump of a transected nerve are a temporal impediment to regeneration in the PNS before Wallerian degeneration rapidly degrades proteoglycans in the extracellular matrix (Hughes et al., 2002) and clears myelin debris (Fu and Gordon, 1997). However, the onset of Wallerian degeneration is slowed by the delayed entry of macrophages into the distal nerve stump (Bruck, 1997). We provide evidence here that overcoming the inhibitory effects of

Acknowledgments

This work was supported by Canadian Institute for Health Research (CHIR) (T.G.) and by the National Institute of Health (T.M.B.). E.U. was recipient of a postdoctoral fellowship from the Spanish ministry of Sciences. The authors thank Guillermo García-Alías for helpful support with the immunohistochemistry.

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