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.

Transplantation of autologous dorsal root ganglia into the peroneal nerve of adult rats: uni- and bidirectional axonal regrowth from the grafted DRG neurons.

Previous studies have demonstrated that transplanted dorsal root ganglion neurons (DRGNs) can survive and differentiate in a variety of orthotopic and heterotopic locations. In order to develop strategies aimed at restoring the sensory function following traumatic injury to the spinal cord and to its peripheral sensory connections, we have transplanted adult autologous dorsal root ganglia (DRGs) into the peroneal nerve of adult rats. Twelve female Sprague-Dawley rats were used. A segment of the peroneal nerve was isolated by double transection and ligature to prevent undesirable reinnervation. The left fifth cervical (C5) DRG was removed from its normal location and inserted into the midportion of the isolated nerve segment. One month after the grafting procedure, a morphological study included axonal retrograde labeling with True Blue (TB) and/or Diamidino Yellow (DY) applied on each cut end of the nerve segment, cell counting, and cell measurement after staining with cresyl violet. Compared to the C5 ganglion maintained in situ, the mean number of surviving DRGNs in the transplant was 1381, corresponding to a survival rate of 20%. Both singly (TB or DY) and doubly (TB + DY) stained DRGNs were encountered. The proportion of surviving neurons that appeared to be doubly labeled was 23%. These neurons were considered as having grown two opposite axonal projections, one into the "central" part of the nerve segment and a second one into its "peripheral" part. The present results give new insights and interesting prospects concerning the possibilities of reconstructing the sensory circuitry after central and/or peripheral injuries.

Estimation of the number and size of female adult rat C4, C5 and C6 dorsal root ganglia (DRG) neurons.

In previous studies primary sensory neurons of adult rats have been counted in lumbar dorsal root ganglia. However, different counting methods have given very different results and at the cervical level, recent data are scarce. In the present study, the number of neurons in C4, C5 and C6 adult rat ganglia was determined using two previously calibrated techniques. The stereological tool was preferred because it directly identifies neurons instead of nucleoli and is more efficient. The C4, C5 and C6 dorsal root ganglia were found to contain 7508+/-299, 6825+/-950 and 6858+/-923 neurons, respectively, and statistical analysis indicated that there was no significant difference between the three levels. There was, however, a great interindividual variation, which was also found at other levels of the spinal cord. The mean diameter of neurons in the C4, C5 and C6 dorsal root ganglia was determined and was 17.52, 20.16 and 20.68 microm, respectively. It is important to know more about the organization of the sensory systems in the normal rat. Once established, the number of neurons in these dorsal root ganglia could be compared with different pathological situations or experimental treatments such as developmental conditions, nerve section or ganglion transplantation.

[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.

Co-transplantation of fetal or adult dorsal root ganglia and of autologous peripheral nerve segments to the adult rat spinal cord: extensive reinnervation of the grafted nerves by the transplanted DRG cells.

Intraspinal transplantation of embryonic neurons and of autologous peripheral nerve segments is an essential tool for studying plasticity and repair in the adult mammalian spinal cord. Unlike adult central nervous system neurons, adult dorsal root ganglion (DRG) cells can be cultured in vitro and are assumed to survive transplantation. In the present work, we have co-transplanted adult (and also fetal, for comparison) DRG and peripheral nerve autografts to the cervical spinal cord of the adult rat. Similar results were obtained from both series: fetal as well as adult DRG cells did survive transplantation and nearly half of them grew lengthy axons into the grafted nerves. A few of them were seen to express a calcitonin gene-related peptide. Possibilities of central afferentation as well as of peripheral connectivity of these transplanted neurons is under study.

Co-transplantation of embryonic neural tissue and autologous peripheral nerve segments to severe spinal cord injury of the adult rat. Guided axogenesis from transplanted neurons.

The present study is the first of a series of experiments designed to investigate the possibilities of reconstructing the severely injured spinal cord by means of transplantation techniques. Special attention has been given here to the capability of transplanted embryonic neurons to extend axons into autologous peripheral nerve grafts (PNGs). A cavity, made unilaterally in the cervical enlargement of the spinal cord of adult rats, was filled with solid pieces of different embryonic tissues: spinal cord (SC), cortex (CT) or dorsal root ganglia (DRG). In more than half of the transplanted animals, one end of a PNG was inserted into the center of the transplants, while the other, extraspinal end, was crushed and tied to peripheral tissues. After a postgrafting period ranging from 1 to 6 months, we found that the 3 types of transplants in general had survived and become integrated with the host spinal cord, although their overall organization remained atypical. Surviving graft neurons had developed processes, some of which had become myelinated. The ability of the grafted neurons to extend axons into the PNG differed strikingly from one type of graft to another, being apparently non-existent for cortical grafts, moderate for spinal cord grafts and quite extensive for dorsal root ganglia transplants. Interestingly, these differences reflected what was observed for the corresponding, fully differentiated qeurons in adult animals, when their cut axons were put in contact with non-neuronal components of peripheral nerves.

Identification of spinal motoneurones in the weakly electric fish, Eigenmannia virescens.

The motoneurones of the spinal cord in the weakly electric fish, Eigenmannia virescens, were investigated by light microscopical observations. For identification of motoneurones HRP injections were made: (1) in the electric organ, (2) in different parts of the trunk muscle, and (3) in the muscle of the anal fin. When the spinal cord was considered in transverse section: (1) labelled cells (EMN), 20-25 micron, were observed in the first case in the dorsal grey between the central canal and dorsal border of the spinal cord; (2) in the second case, labelled cells (MN1), 35-40 micron, were found in the central grey ventro-laterally, in close vicinity of the ependymal canal; (3) in the last case, labelled cells (MN2), 18-20 micron, were seen in the ventro-lateral part of the anterior horn. When the spinal cord was considered in longitudinal sections, EMNs and MN1 constitute a continuous column along the spinal axis, whereas MN2 show a metametric distribution. The 3 groups of motor cells can be identified in the spinal cord for the whole length of the anal fin, whereas the MN1 and MN2 disappear at the level of the caudal peduncle. The 3 cell types bear dendritic processes; the EMN thin ones and the MN1 and 2 rather large ones. The results indicate that distinct cell groups innervate the electric organ (EMN), the large muscles of the trunk (MN1) and those of the anal fin (MN2).