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Inhibition of caspases promotes long-term survival and reinnervation by axotomized spinal motoneurons of denervated muscle in newborn rats

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Abstract

We examined whether (1) a pan-caspase inhibitor, Boc-D-FMK, exerts long-term neuroprotective effects on spinal motoneurons (MNs) after root avulsion in neonatal rats and (2) whether the rescued spinal MNs regenerate their axons into a peripheral nerve (PN) graft and reinnervate a previously denervated target muscle. Eight weeks after root avulsion, 67% of spinal MNs remained in the Boc-D-FMK-treated group, whereas all MNs died in the sham control group. By 12 weeks postinjury, however, all Boc-D-FMK treated MNs died. In the regeneration experiment, a PN graft was implanted at different times after injury. The animals were allowed to survive for 4 weeks following the operation. Without caspase inhibition, MNs did not regenerate at any time point. In animals treated with Ac-DEVD-CHO, a caspase-3-specific inhibitor, and Boc-D-FMK, 44 and 62% of MNs, respectively, were found to regenerate their axons into a PN graft implanted immediately after root avulsion. When the PN graft was implanted 2 weeks after injury, however, MNs failed to regenerate following Ac-DEVD-CHO treatment, whereas 53% of MNs regenerated their axons into the graft after treatment with Boc-D-FMK. No regeneration was observed when a PN graft was implanted later than 2 weeks after injury. In the reinnervation study, injured MNs and the target biceps muscle were reconnected by a PN bridge implanted 2 weeks after root avulsion with administration of Boc-D-FMK. Eight weeks following the operation, 39% of MNs reinnervated the biceps muscle. Morphologically normal synapses and motor endplates were reformed in the muscle fibers. Collectively, these data provide evidence that injured neonatal motoneurons can survive and reinnervate peripheral muscle targets following inhibition of caspases.

Introduction

Brachial plexus injury leads to massive MN degeneration. Since the target muscle lacks innervation, muscular atrophy occurs and therefore some patients lose part or all of their arm movement. Every year many infants suffer from this injury when they are delivered improperly Hoeksma et al 2000, Noetzel et al 2001, Bar et al 2001. A recent clinical strategy for the treatment of brachial plexus injury is to reimplant the avulsed root into the lesioned area Carlstedt et al 2000, Fournier et al 2001. However, surgical difficulties, determining an optimum site for reimplantation and the patients age all affect the outcome of recovery Jamieson and Hughes 1980, Fournier et al 2001. Therefore, it is crucial to develop an effective treatment for pediatric brachial plexus injury.

The age of the animal and the type of injury are two important factors that affect the fate of spinal MNs after axonal injury. In adult rats, the majority of spinal MNs survive following distal axotomy, whereas most of them die after root avulsion (Wu, 1993). In developing animals, by contrast, almost all MNs die after either distal axotomy or root avulsion Yuan et al 2000, Chan et al 2001. Age also affects the regenerative capacity of spinal MNs after axonal injury. It has been shown that axonal regrowth into a PN graft is possible for adult CNS neurons Sorbie and Porter 1969, David and Aguayo 1981, David and Aguayo 1985, Richardson et al 1982, Richardson et al 1984, Horvat 1991, Emery et al 1997 and following root avulsion in adult rats, spinal MNs are able to regenerate their axons into a reimplanted PN graft Wu et al 1994, Chai et al 2000, whereas neonatal MNs are unable to regenerate into an implanted PN graft (Chan et al., 2002). The failure to regenerate in neonates may be due to (1) MN death before any regeneration occurs since virtually all spinal MNs degenerate within 7 days following root avulsion or (2) developing MNs may lose the capacity for regeneration after axonal injury. We have previously shown that caspase inhibitors increase the survival rate of neonatal spinal MNs for up to 3 weeks following root avulsion (Chan et al., 2001), which may provide a larger window for examining the regenerative capacity of injured developing MNs. It has been shown in adult animals that injured spinal MNs can regenerate through a PN bridge Emery et al 2000, Rhrich-Haddout et al 2001 and form functional synaptic contacts Horvat et al 1989, Pecot-Dechavassine and Mira 1994. The PN bridge consists of Schwann cells and extracellular molecules and both may play crucial roles in axonal regeneration Villegas-Perez et al 1988, Carbonetto 1991, Morrissey et al 1991, Guenard et al 1992, Bunge 1994. However, whether spinal MNs can reinnervate target muscles and form functional connections through a PN bridge in neonates remains unclear.

Although the precise mechanism for MN death after axonal injury is not fully understood, many reports have shown that such death may be triggered by an apoptotic pathway Rothstein et al 1994, Yoshiyama et al 1994, Lo et al 1995, Oliveira et al 1997, Li et al 1998, Martin 1999, Blondet et al 2001, Liu and Martin 2001a, Liu and Martin 2001b. Caspases are important mediators of apoptosis Milligan et al 1995, Barnes et al 1998, Hayashi et al 1998, Turgeon et al 1998 and inhibition of various kinds of caspases can significantly promote the survival of CNS neurons after injury. Peptide inhibitors of caspases arrest apoptotic cell death of MNs in both in vivo and in vitro models Milligan et al 1995, Allen et al 2001, Gerhardt et al 2001, Hayashi et al 2001, Von Coelln et al 2001. We have previously found that a single dose of the pan-caspase inhibitor benzyloxycarbonyl-Asp(OMe)fluoromethylketone (Boc-D-FMK) rescued more than 70% of MNs for at least 3 weeks after root avulsion (Chan et al., 2001). Although the caspase-3-specific inhibitor N-acetyl-Asp-Glu-Val-Asp aldehyde (Ac-DEVD-CHO) also promoted MN survival in this situation, the effect was lost by 2 weeks postinjury.

Section snippets

Surgical procedures

On the day of birth, newborn female Spraque–Dawley rats were anesthetized under deep hypothermia. Under a surgical microscope, a dorsal laminectomy was carried out and the spinal root of the seventh cervical (C7) segment was identified. The C7 ventral root together with the dorsal root were avulsed by a pair of microhemostatic forceps.

To study the long-term neuroprotective effect of Boc-D-FMK, animals were divided into two groups. There were six rats in each group at each time point. The first

Long-term neuroprotective effect of Boc-D-FMK

Motoneurons were identified and counted as described previously (Clarke and Oppenheim, 1995). In brief, only MNs with a large nucleus containing clearly visible nucleoli and a largely distinct cytoplasm were counted. Because the number of MNs on the contralateral intact side of the experimental animals was not significantly different from normal control animals (data not shown), the contralateral side served as an internal control. We have previously reported that by 7 days postlesion, there

Discussion

The present results indicate that caspases play a key role in the death of spinal MNs after injury in neonates. Inhibition of caspases led to long-term neuroprotection as well as axonal regeneration of avulsed spinal MNs. With a PN bridge between the spinal cord and the denervated muscle target, the caspase inhibitor-treated MNs were able to reinnervate the neuromuscular junction and muscular atrophy was reduced. These results suggest that the inhibition of caspases may be a potent strategy for

Conclusion

The experiments presented here provide evidence that following root avulsion, neonatal spinal MNs can survive and reinnervate target muscle if appropriate treatment is provided. A single injection of Boc-D-FMK results in long-term protection of MNs against root avulsion-induced death for more than 8 weeks and the Boc-D-FMK-treated MNs are able to regenerate their axons into an implanted PN graft and reinnervate the target muscle. Taken together, these data suggest that local administration of

Acknowledgements

This study was supported by research grants from the University of Hong Kong and the Hong Kong Research Grants Council.

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