Reinnervation of a denervated skeletal muscle by spinal axons regenerating through a collagen channel directly implanted into the rat spinal cord.

In the present study, the continuity between the central nervous system (CNS) and the peripheral nervous system (PNS) was restored by mean of a collagen channel in order to reinnervate a skeletal muscle. Three groups of animals were considered. In the first group, one end of the collagen channel was implanted in the cervical spinal cord of adult rats. The other end was connected to a 30-mm autologous peripheral nerve graft (PNG) implanted into the denervated biceps brachii muscle. The gap between the spinal cord and the proximal nerve stump varied from 3 to 7 mm. In the second group of animals, the distal end of the PNG graft was ligatured in order to compare the survival of the growing axons in the presence and in the absence of a muscular target. In the third group of animals, the extraspinal stump of the collagen channel was ligatured. Our study demonstrates that spinal neurons and dorsal root ganglion (DRG) neurons can grow long axons through the collagen channel over a 7-mm gap and reinnervate a denervated skeletal muscle. The results also indicate that the presence of a PNG at the extraspinal stump of the collagen channel is essential for axonal regrowth and that the muscle target contributes to the long-term maintenance of the regenerating axons. These data might be interesting for clinical application when the continuity between the CNS and PNS is interrupted such as in root avulsion.

Alpha-motoneurons of the injured cervical spinal cord of the adult rat can reinnervate the biceps brachii muscle by regenerating axons through peripheral nerve bridges: combined ultrastructural and retrograde axonal tracing study.

Following our previous studies related to brachial plexus injury and repair, the present experimentation was designed to examine the ultrastructural features of those motoneurons of the locally injured cervical spinal cord of adult rats that were seen to regenerate into peripheral nerve (PN) bridges and to reinnervate nearby skeletal muscles. Here, the peripheral connection of the PN bridge was made with the biceps brachii (BB) muscle. Three months postsurgery, the spinal motoneurons labelled by retrograde axonal transport of horseradish peroxidase (HRP), after its injection into the BB, were selected on thick sections, using light microscopy, for the presence of dark amorphous granules of the HRP reaction product. Serial ultrathin sections were then made from the selected material. For the 10 labelled neurons studied, we examined the synaptic boutons present on the membrane of the neuronal soma. For five of them, we could observe three of the six types of synaptic boutons described for the alpha-motoneurons of the cat (S-type with spherical vesicles, F-types with flattened vesicles, and C-type with subsynaptic cistern). The largest boutons (type C) are specific to alpha-motoneurons. In comparison to normal material, we noticed a decrease in the number of boutons and an increase in the number of glial processes. After a transient phase of trophic changes, the reinnervated BB muscles showed a return of their fibers to nearly normal diameters as well as evidence of fiber type grouping. Simultaneous staining with silver and cholinesterase also revealed the presence of new motor endplates frequently contacted by several motoneurons. The present study indicates that, after a local spinal injury, typical alpha-motoneurons can reinnervate a skeletal muscle by regenerating axons into the permissive microenvironment provided by a PN graft. These data offer prospects for clinical reconstruction of the brachial plexus after avulsion of one or several nerve roots.

Motoneurons of the adult marmoset can grow axons and reform motor endplates through a peripheral nerve bridge joining the locally injured cervical spinal cord to the denervated biceps brachii muscle.

Reconnection of the injured spinal cord (SC) of the marmoset with the denervated biceps brachii muscle (BB) was obtained by using a peripheral nerve (PN) bridge. In 13 adult males, a 45 mm segment of the peroneal nerve was removed: one end was implanted unilaterally into the cervical SC of the same animal (autograft), determining a local injury, although the other end was either directly inserted into the BB (Group A) or, alternatively, sutured to its transected motor nerve, the musculocutaneous nerve (Group B). From 2-4 months post-surgery, eight out of the 10 surviving animals responded by a contraction of the BB to electrical stimulations of the PN bridge. All ten were then processed for a morphological study. As documented by retrograde axonal tracing studies using horse radish peroxidase or Fast Blue (FB), a mean number of 314 (Group A) or 45 (Group B) spinal neurons, mainly located close to the site of injury and grafting, re-expressed a capacity to grow and extend axons into the PN bridge. Most of these regenerated axons were able to grow up to the BB and form or reform functional motor endplates. Many of the spinal neurons that were retrogradely labeled with FB simultaneously displayed immunoreactivity for choline acetyl-transferase and consequently were assumed to be motoneurons. Reinnervation and regeneration of the BB were documented by methods revealing axon terminals, endplates and myofibrillary ATPase activity. Our results indicate that motoneurons of the focally injured SC of a small-sized primate can, following the example of the adult rat, re-establish a lost motor function by extending new axons all the way through a PN bridge connected to a denervated skeletal muscle.

Expression of peripherin, NADPH-diaphorase and NOS in the adult rat neocortex.

Peripherin is mainly expressed in peripheral neurones and in CNS neurones which extend axons into peripheral nerves. However, this intermediate filament protein has also been detected in a few other neurones entirely located within the CNS. The present study focuses on the adult rat neocortex. Peripherin immunoreactive (P+) neuronal somata and their neuritic extensions were observed in cortical layers II, III, V and VI, while a few P+ nerve fibres could be seen in layer I. All the P+ neurones could be selectively stained using reduced nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) histochemistry, a typical feature of aspiny neurones. Some of the P+ neurones could also be immunostained with an antibody raised against nitric oxide synthase (NOS). These results provide evidence that peripherin is present in a discrete population of aspiny interneurones of the adult rat neocortex. The functional significance of the co-expression of peripherin and NOS needs further investigation.

[Post-traumatic reconnection of the cervical spinal cord with skeletal striated muscles. Study in adult rats and marmosets]. / Reconnexion post-traumatique de la moelle épinière cervicale avec la musculature striée squelettique. Etude chez le rat et le marmouset adultes.

In an attempt at repairing the injured spinal cord of adult mammals (rat, dog and marmoset) and its damaged muscular connections, we are currently using: 1) peripheral nerve autografts (PNG), containing Schwann cells, to trigger and direct axonal regrowth from host and/or transplanted motoneurons towards denervated muscular targets; 2) foetal spinal cord transplants to replace lost neurons. In adult rats and marmosets, a PNG bridge was used to joint the injured cervical spinal cord to a denervated skeletal muscle (longissimus atlantis [rat] or biceps brachii [rat and marmoset]). The spinal lesion was obtained by the implantation procedure of the PNG. After a post-operative delay ranging from 2 to 22 months, the animals were checked electrophysiologically for functional muscular reconnection and processed for a morphological study including retrograde axonal tracing (HRP, Fast Blue, True Blue), histochemistry (AChE, ATPase), immunocytochemistry (ChAT) and EM. It was thus demonstrated that host motoneurons of the cervical enlargement could extend axons all the way through the PNG bridge as: a) in anaesthetized animals, contraction of the reconnected muscle could be obtained by electrical stimulation of the grafted nerve; b) the retrograde axonal tracing studies indicated that a great number of host cervical neurons extended axons into the PNG bridge up to the muscle; c) many of them were assumed to be motoneurons (double labelling with True Blue and an antibody against ChAT); and even alpha-motoneurons (type C axosomatic synapses in HRP labelled neurons seen in EM in the rat); d) numerous ectopic endplates were seen around the intramuscular tip of the PNG. In larger (cavitation) spinal lesions (rat), foetal motoneurons contained in E14 spinal cord transplants could similarly grow axons through PNG bridges up to the reconnected muscle. Taking all these data into account, it can be concluded that neural transplants are interesting tools for evaluating both the plasticity and the repair capacities of the mammalian spinal cord and of its muscular connections.

Expression of peripherin in solid transplants of foetal spinal cord and dorsal root ganglia grafted to the injured cervical spinal cord of adult rats.

The expression of the neuronal type III intermediate filament protein peripherin was studied in E14 spinal cord fragments and E15 dorsal root ganglia 1-30 weeks after their transplantation to the injured cervical spinal cord of the adult rat. In the dorsal root ganglion transplants, the surviving neurons generally appeared as a rather healthy looking population of small strongly immunoreactive cells which are very similar to the small dorsal root ganglion neurons of adult control rats. In the spinal cord transplants, there were only a few peripherin-immunoreactive neurons, morphologically close to the motoneurons or to the preganglionic sympathetic neurons of adult rats. In both types of transplants, peripherin expression of the immunoreactive neurons was apparently correlated with the previously established ability of these transplanted neurons for extensive axonal growth into a co-grafted peripheral nerve.