A spatio-temporal analysis of motoneuron survival, axonal regeneration and neurotrophic factor expression after lumbar ventral root avulsion and implantation
Introduction
Injury to spinal nerve roots in humans occurs most frequently following traffic accidents or during complicated child birth. The majority of lesions are located in the brachial plexus. These injuries are complex and regeneration and recovery of function is poor, especially when an avulsion of a spinal root occurs directly at the spinal cord surface. Following root avulsion, the absence of a proximal nerve stump precludes reconstructive surgery of the spinal nerves. In patients, rerouting of the intercostal nerve towards the distal target of the avulsed root (Malessy and Thomeer, 1998), or direct reimplantation of the avulsed root into the affected spinal cord segment (Carlstedt et al., 2000, Cullheim et al., 1999, Carlstedt et al., 1995) have resulted in some recovery of function. However, following reimplantation, reinnervation of distal targets is poor as indicated by decreasing muscle force in distal direction (Carlstedt, 2008). Avulsion of ventral roots in experimental animals results in severe spinal motoneuron atrophy and death occurs within weeks after the lesion (Koliatsos et al., 1994, Novikov et al., 1995, Wu and Li, 1993). Reimplantation of the avulsed ventral root has beneficial effects on the injured motoneurons and attracts regenerating fibers over several spinal cord segments resulting in a delay of motoneuron degeneration (Chai et al., 2000, Hoang and Havton, 2005). Following axotomy, Schwann cells in denervated distal nerve segments proliferate and display increased production of neurotrophic factors (Meyer et al., 1992, Hoke et al., 2000, Hammarberg et al., 1996, Naveilhan et al., 1997, Funakoshi et al., 1993). The neurotrophic factors produced by the Schwann cells in the reimplanted roots have been suggested to create a favorable environment for regeneration, and the beneficial effect of reimplantation of avulsed spinal nerve roots has therefore been studied in several models (Hoang et al., 2006a, Jivan et al., 2006, Wu et al., 1994).
Following the avulsion and reimplantation of ventral roots innervating the forepaw or bladder, without further treatment successful recovery of function has been reported in a number of studies (Gu et al., 2004, Hoang et al., 2006b). In contrast, avulsion and reimplantation of the lumbar ventral nerve roots innervating the hind limb has resulted in fiber ingrowth into the implanted ventral root but poor distal regeneration, although reimplantation combined with riluzole and GDNF resulted in limited improvement of hind limb function (Nogradi and Vrbova, 2001, Bergerot et al., 2004, Blits et al., 2004, Eggers et al., 2008). A likely explanation for the difference in recovery following reimplantation of cervical and sacral ventral roots as compared to lumbar roots could be the substantially longer distance axons have to travel towards the target muscles following lumbar ventral root avulsion. This explanation has been suggested previously as a potential reason of poor function recovery in humans (Hoke, 2006, Gordon et al., 2003). After prolonged periods of nerve denervation, the injured nerve atrophies and breakdown of basement membrane and loss of bands of Bungner occurs (Giannini and Dyck, 1990, Vuorinen et al., 1995). As frequently discussed, prolonged denervation of distal nerve and target, as well as prolonged axotomy strongly reduces the success of axonal regeneration and target reinnervation (Gordon et al., 2003, Bergerot et al., 2004, Hoke et al., 2006, Jivan et al., 2006). The spatio-temporal pattern of motor axon growth towards their distal target following lumbar spinal nerve avulsion and reimplantation has not been studied in great detail.
The current experiment was designed, to determine whether the absence of functional recovery following lumbar ventral root reimplantation is indeed the result of poor long-distance regeneration of motor axons into the distal portion of the sciatic nerve. We evaluated motoneuron survival and regeneration of motor axons into the implanted root, lumbar plexus and sciatic nerve at early (1 and 2 weeks), intermediate (4 and 8 weeks) and long (16 weeks) time points after reimplantation. Furthermore, the temporal expression profile of 4 neurotrophic factors in the distal sciatic nerve and the extent of hind limb target muscle atrophy were investigated. We show that implantation of avulsed ventral roots prolongs motoneuron survival up to 4 weeks. Numerous motoneuron fibers enter the implanted roots and the lumbar plexus, but many fibers fail to regenerate towards the distal sciatic nerve. At later time points a gradual decline of distal neurotrophic support and target muscle weight was observed. These findings suggest that regeneration of motor axons and recovery of function following reimplantation of avulsed lumbar ventral roots is hindered by prolonged denervation of the distal nerve and severe atrophy of the hind limb muscles.
Section snippets
Experimental animals and surgical procedure
A total of 50 female Wistar rats (200–250 g; Harlan, Horst, The Netherlands) was used in this study. Animals were housed under standard conditions at a 12:12 h light/dark cycle with food and water ad libitum. Experimental procedures were performed in accordance with the European guidelines for the care and use of laboratory animals (86\609\EEC) and were approved by the animal experimentation committee of the Royal Netherlands Academy of Sciences. Animals were randomly assigned to either an
Implantation of avulsed ventral roots promotes motoneuron survival
To quantify the degree of motoneuron survival after ventral root avulsion and implantation, serial transverse spinal cord sections were cut of all animals at 1, 2, 4, 8 and 16 weeks post-lesion. Motoneurons were stained for ChAT and subsequently motoneuron profiles were counted. At all post-lesion time points, the number of motoneurons on the contralateral side was unaffected and displayed a normal morphology (Fig. 2A). ChAT immunoreactivity was slightly reduced in the affected motoneurons
Discussion
The results of the present study are summarized in Fig. 9 and show that: i. Reimplantation of avulsed lumbar ventral roots immediately after avulsion prolongs the survival of motoneurons with approximately 4 weeks, ii. Numerous growth cones and regenerating motor axons have entered the ventral root during the first 2 weeks post-lesion and continue to extend into the lumbar plexus during the subsequent weeks, iii. Some axons continue to grow more distally into the sciatic nerve resulting in a
Acknowledgments
The authors would like to thank Joop J.v. Heerikhuize for his excellent technical assistance with nerve fiber quantification procedures. This work was partially supported by SenterNovem (project number: ISO52022).
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