M. Risling

Motoneurons reinnervate skeletal muscle after ventral root implantation into the spinal cord of the cat

By use of intracellular recording and staining with horseradish peroxidase it was found that alpha and probably also gamma motoneurons were able to reinnervate ventral root implants after an avulsion of ventral roots at the spinal cord surface in the cat. The reinnervation of the implant was achieved after an initial growth of new axons in central nervous system tissue. Reinnervating neurons could be excited or inhibited by segmental reflex activity and their axons could conduct nerve impulses. The character of muscle twitch responses elicited by electrical stimulation of implanted roots strongly indicated that denervated muscles were reinnervated by new motor axons via the implant.

Peripheral nerve section induces increased levels of calcitonin gene-related peptide (CGRP)-like immunoreactivity in axotomized motoneurons

SummaryBy use of fluorescence immunohistochemistry it is shown that sciatic nerve section in cat and rat induces increased levels of immunoreactive calcitonin gene-related peptide (CGRP) in axotomized motoneurons. In the rat, this effect was clearly seen at 2–5 days postoperatively, but could not be demonstrated after 11–21 days. These findings are discussed in relation to previously proposed roles for CGRP in motoneurons.

‘Dendraxons’ in regenerating motoneurons in the cat: do dendrites generate new axons after central axotomy?

The intramedullary portions of motor axons in the spinal cord of adult cats were divided by longitudinal incisions in the ventral funiculus. After 7-11 weeks ventral horn neurons were injected intracellularly with horseradish peroxidase. Regenerating processes of the injected cells were studied with light- and electron-microscopical techniques. The results show that in some cases more than one axon-like myelinated regenerating process was found in a single neuron. Moreover, in such cases at least one of the processes seemed to be of dendritic origin.

Reinnervation of the ventral root L7 from ventral horn neurons following intramedullary axotomy in adult cats

Through small longitudinal incisions in the ventral funiculus of the adult cat spinal cord the intramedullary portions of motoraxons forming the L7 ventral root were divided. The animals were sacrificed by glutaraldehyde-perfusion 1-15 weeks postoperatively. In some cases this was preceded by HRP injection into large L7 ventral horn neurons. Sections from the lesioned ventral funiculus and the denervated ventral root L7 were examined by light and electron microscopy. During the first postoperative month increasing numbers of unmyelinated and thinly myelinated large-diameter axons coursed through the lesion and entered the denervated ventral root. Occasional neuroma-like formations were also found in the lesion area. After 5 weeks survival clusters of regenerating axons reached the distal end of the L7 ventral root. Light microscopic examination of Vibratome sections from HRP-injected animals showed that at least some regenerated axons come from large neurons, presumably alpha-motoneurons, in the ventral horn motor nuclei. Most of the examined labeled regenerated axons reached the CNS/PNS junction of the ventral root but a few followed a grossly aberrant course and/or terminated within the lesion. These observations show that regeneration of large-diameter axons through a CNS-lesion is possible. Both the intrinsic regenerative capacity of the axotomized neurons and the presence of a denervated ventral root in the immediate vicinity are likely to be contributing factors with respect to this example of successful CNS axon regeneration.

Effects of sciatic nerve resection on L7 spinal roots and dorsal root ganglia in adult cats

The size, distribution, and number of nerve fibers and neuronal perikarya in the L7 spinal roots and ganglia of adult cats were examined 35, 90, and 190 days after ipsilateral sciatic nerve resection. With increasing survival time the size spectra of myelinated ventral root nerve fibers showed a progressive flattening of the alpha peak. In the dorsal roots the myelinated fiber size distribution exhibited a marked shift toward smaller sizes. The reduction in the proportion of large myelinated axons was particularly evident in the dorsal roots. Less clearcut changes were found in the size distribution of spinal ganglion neuronal perikarya. No significant loss of axons could be detected in ventral or dorsal roots. There was, however, a marked reduction in the number of dorsal root ganglion neurons. This discrepancy suggested the possibility that an initial loss of dorsal root axons was concealed by recurrent sprouting of axons from the proximal nerve stump. However, neuroma excision 90 days after nerve resection did not lead to any reduction in dorsal root axon numbers. Thus, any ingrowth of new axons to the dorsal root should occur from levels proximal to the neuroma. In comparison with previous findings in kittens, peripheral nerve resection in adult cats had significantly smaller effects on sizes and numbers of spinal root nerve fibers as well as of dorsal root ganglion neurons. Therefore, the potential for restitution of the peripheral innervation by axon regeneration appeared to be basically greater in mature than in immature animals.

Expression of GAP-43 mRNA in the adult mammalian spinal cord under normal conditions and after different types of lesions, with special reference to motoneurons

SummaryIn situ hybridization histochemistry was used to detect cell bodies expressing mRNA encoding for the phosphoprotein GAP-43 in the lumbosacral spinal cord of the adult rat, cat and monkey under normal conditions and, in the cat and rat, also after different types of lesions. In the normal spinal cord, a large number of neurons throughout the spinal cord gray matter were found to express GAP-43 mRNA. All neurons, both large and small, in the motor nucleus (Rexeds lamina IX) appeared labeled, indicating that both alpha and gamma motoneurons express GAP-43 mRNA under normal conditions. After axotomy by an incision in the ventral funiculus or a transection of ventral roots or peripheral nerves, GAP-43 mRNA was clearly upregulated in axotomized motoneurons, including both alpha and gamma motoneurons. An increase in GAP-43 mRNA expression was already detectable 24 h postoperatively in lumbar motoneurons both after a transection of the sciatic nerve at knee level and after a transection of ventral roots. At this time, a stronger response was seen in the motoneurons which had been subjected to the distal sciatic nerve transection than was apparent for the more proximal ventral root lesion. An upregulation of GAP-43 mRNA could also be found in intact motoneurons located on the side contralateral to the lesion, but only after a peripheral nerve transection, indicating that the concomitant influence of dorsal root afferents may play a role in GAP-43 mRNA regulation. However, a dorsal root transection alone did not seem to have any detectable influence on the expression of GAP-43 mRNA in spinal motoneurons, while the neurons located in the superficial laminae of the dorsal horn responded with an upregulation of GAP-43 mRNA. The presence of high levels of GAP-43 in neurons has been correlated with periods of axonal growth during both development and regeneration. The role for GAP-43 in neurons under normal conditions is not clear, but it may be linked with events underlying remodelling of synaptic relationships or transmitter release. Our findings provide an anatomical substrate to support such a hypothesis in the normal spinal cord, and indicate a potential role for GAP-43 in axon regeneration of the motoneurons, since GAP-43 mRNA levels was strongly upregulated following both peripheral axotomy and axotomy within the spinal cord. The upregulation of GAP-43 mRNA found in contralateral, presumably uninjured motoneurons after peripheral nerve transection, as well as in dorsal horn neurons after a dorsal root transection, indicates that GAP-43 levels are altered not only as a direct consequence of a lesion, but also after changes in the synaptic input to the neurons.

Spinal axons in central nervous system scar tissue are closely related to laminin-immunoreactive astrocytes

Although transected central nervous system axons fail to regrow after injuries in adult mammals, they send sprouts into the scar tissue that forms at the lesion. We have investigated the relation between scar cells, laminin-like immunoreactivity and cut spinal axons in two previously characterized spinal cord lesion types. Labeling with antisera to glial fibrillary acidic protein and laminin demonstrated that the scar tissue formed after lesions in the rat and cat dorsal and ventral funiculi showed prominent gliosis and strong laminin-like immunoreactivity four days to one year postlesion. Axonal sprouts in the scar, visualized with antibodies to neurofilament (RT97) or by tracing using fluorescein-conjugated dextran, were ensheathed by a thin layer of strongly laminin-immunoreactive tissue. Immunoelectron microscopy demonstrated that axons in the scar were ensheathed predominantly by astrocytes, and that the surface of the cells outlining the axons in the scar showed strong laminin-like immunoreactivity. Adhesive and neurite orienting properties in the scar tissue were assessed in an in vitro system where PC12 cells were cultured on spinal cord slices from dorsal funiculus-lesioned rats. Very few cells adhered to the spinal cord section except for the part where the scar tissue had formed, where numerous cells were attached. The PC12 cells that had adhered to the scar tissue were mainly seen in parts of the scar that showed laminin-like immunoreactivity and their neurites predominantly followed tissue showing laminin-like immunoreactivity. The close association between axonal sprouts and laminin-like immunoreactivity indicates a role for laminin in axonal growth and/or guidance in the injured spinal cord.

Spinal cord implantation of avulsed ventral roots in primates; correlation between restored motor function and morphology

Abstract Functional restitution following spinal cord implantation of avulsed ventral roots was assessed electromyographically and correlated with the morphology of the regenerated neural structures in primates. The C5–C8 ventral roots were avulsed from the spinal cord in seven Macaca fascicularis monkeys. In three animals the roots were immediately reimplanted into the ventrolateral part of the spinal cord. In two monkeys the avulsed roots were reimplanted with a delay of 2 months and in two control animals the roots were not reimplanted. There was substantial recovery of function after both immediate and delayed spinal cord implantation of the avulsed ventral roots. The population of neurons that had regenerated was larger than on the control side, indicating a rescue of cells after an immediate root implantation. Different functional types of neurons had been attracted to regrow axons to the implanted root as judged by their position in the ventral horn. Thus, neurons normally supplying antagonistic muscles, such as the triceps muscle, participated in the innervation of the biceps muscle. Functionally this deficient directional specificity was correlated to both spasticity and co-contractions among agonistic and antagonistic muscles. Occasional electromyographic signs of function occurred also in control animals where the avulsed roots had not been implanted. This recovery was found to depend on regrowth from the site of avulsion, within the pia mater among the leptomeningeal cells and to the avulsed roots. The acceptable functional dexterity regained due to corrective surgery is discussed in terms of neurotrophism and plasticity.

Regrowth of motor axons following spinal cord lesions: Distribution of laminin and collagen in the CNS scar tissue

In previous studies we have demonstrated that spinal motoneurons in the adult cat can regenerate CNS-type axons through CNS scar tissue into denervated ventral roots. This scar tissue, which appears to support and sustain the growth of injured CNS axons, has been shown to have a persistent defect in the blood-brain barrier (BBB). In the present study, the binding of antibodies to nerve growth factor receptor (NGFr), laminin, collagen, and a microtubule associated protein (MAP5) was assessed with indirect immunohistochemical methods 4 days-20 weeks after a lesion in the ventral funiculus of the spinal cord. An increase in content of collagen-, laminin-, and NGFr-like immunoreactivity was observed in the scar tissue during the first 3 weeks. Although type I collagen dominated in superficial areas of the scar, type IV collagen and laminin-like immunoreactivity was observed in expanded perivascular spaces all over the lesion zone. Type IV collagen- and laminin-immunoreactive structures sometimes appeared to form strands which interconnected the ventral horn and the ventral root. Regenerating axons, as revealed by staining with MAP5 or NGFr antibodies, were observed in close association to these paths. It has been suggested that a breakdown of the BBB may play a vital role in certain types of CNS regeneration by increasing the access of blood-borne trophic factors to the lesion area. The demonstration of extracellular matrix proteins like laminin provides further evidence for the notion that the observed regenerative growth takes place in an environment that is markedly different from the normal CNS.

Nerve fibre regeneration across the PNS-CNS interface at the root-spinal cord junction.

Root-spinal cord regeneration was investigated in immature and adult rats. The elongation in the dorsal root of regrowing dorsal root axons, rerouted ventral root nerve fibres (cholinergic neurons) or hypogastric nerve fibres (catecholaminergic neurons) is impeded as they meet the astrocyte dominated CNS tissue of the root. The establishment of synaptoid nerve terminals as the regrowing axons encounter astrocytes indicates a mechanism for growth inhibition other than a physical impediment in the CNS environment. The glial cells of the CNS segment in the root are influenced by the type of regenerating nerve fibres in terms of maintenance, multiplication and phenotypic expression. After a dorsal root lesion in the neonatal rat several root axons may reinnervate the spinal cord. In these rats, the normal establishment of a CNS root segment has been disrupted and the PNS-CNS border is situated central to the root-spinal cord junction. Implantation of cut dorsal roots into the spinal cord of adult rats results in the extension of processes from intrinsic spinal cord neurons out into the root. After implantation of avulsed ventral roots into the ventro-lateral aspect of the cord, axonal regrowth and functional restitution of alpha-motoneurons could be demonstrated by intracellular recordings and injections with horseradish peroxidase. These results show that regeneration can occur across a PNS-CNS interface that has been established secondary to a trauma in the mature animal and in the immature animal before the astrocyte-rich CNS root segment has been developed.