Anatomical and functional recovery folloing spinal cord transection in the chick embryo

Following complete transection of the thoracic spinal cord at various times during embryonic development, chick embryos and posthatched animals exhibited various degrees of anatomical and functional recovery depending upon the age of injury. Transection on embryonic day 2 (E2), when neurogenesis is still occurring and before descending or ascending fiber tracts have formed, produced no noticeable behavioral or anatomical deficits. Embryos hatched on their own and were behaviorally indistinguishable from control hatchlings. Similar results were found following transection on E5, an age when neurogenesis is complete and when ascending and descending fiber tracts have begun to project through the thoracic region. Within 48 h following injury on E5, large numbers of nerve fibers were observed growing across the site of transection. By E8, injections of horseradish peroxidase (HRP) administered caudal to the lesion, retrogradely labelled rostral spinal and brainstem neurons. Embryos transected on E5 were able to hatch and could stand and locomote posthatching in a manner that was indistinguishable from controls. Following spinal cord transections on E10, anatomical recovery of the spinal cord at the site of injury was not quite as complete as after E5 transection. Nonetheless, anatomical continuity was restored at the site of injury, axons projected across this region, and rostral spinal and brainstem neurons could be retogradely labelled following HRP injections administered caudal to the lesion. At least part of this anatomical recovery may be mediated by the regeneration or regrowth of lesioned axons. Although none of the embryos transected on E10 that survived to hatching were able to hatch on their own, because several shamoperated embryos were also unable to hatch, we do not attribute this deficit to the spinal transection. When E10-transected embryos were aided in escaping from the shell, they were able to support their own weight, could stand, and locomote, and were generally comparable, behaviorally, to control hatchlings. Repair of the spinal cord following transection on E15 was considerably less complete compared to embryos transected on E2, E5, or E10. However, in some cases, a degree of anatomical continuity was eventually restored and a few spinal neurons rostral to the lesion could be retrogradely labelled with HRP. By contrast, labelled brainstem neurons were never observed following E15 transection. E15 transected embryos were never able to hatch on their own, and when aided in escaping from the shell, the hatchlings were never able to stand, support their own weight or locomote. We conclude that successful anatomical and functional recovery occurs following a complete spinal cord transection in the chick embryo made any time between E2 and E10. By E15, however, there is an altered response to the transection such that anatomical continuity is not restored sufficiently to mediate behavioral or functional recovery. Although the altered response of the chick embryo spinal cord to injury between E10 and E15 could be due to a variety of factors, we favor the notion that cellular or molecular changes associated with axonal growth and guidance occur at this time that are responsible for the transition from successful to unsuccessful recovery.     

Rewired glycosylation activity promotes scarless regeneration and functional recovery in spiny mice after complete spinal cord transection

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Spontaneous synaptic activity in cell culture of chick embryo spinal cord

The spontaneous development of synaptic activity (SSA) was studied in cell cultures of chick embryo spinal cord. The complicated time structure of the SSA, an important early-stage characteristic of which was giant inhibitory postsynaptic currents (IPSC), was demonstrated. The ionic nature and pharmacological sensitivity of these IPSC suggest that glycine is their transmitter. Emergence of excitatory postsynaptic currents (EPSC) and complex antagonistic relationships between excitatory and inhibitory SSA was detected later. Possible mechanisms for maintenance of synaptic activity during the inhibitory function are discussed. Correlations between the regularities of synaptic transmission development that we have disclosed and neuronal circuit electrical activity are examined.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the USSR, Kiev. Translated from Neirofiziologiya, Vol. 23, No. 3, pp. 280–290, May–June, 1991.     

EphA4 blockers promote axonal regeneration and functional recovery following spinal cord injury in mice

Upregulation and activation of developmental axon guidance molecules, such as semaphorins and members of the Eph receptor tyrosine kinase family and their ligands, the ephrins, play a role in the inhibition of axonal regeneration following injury to the central nervous system. Previously we have demonstrated in a knockout model that axonal regeneration following spinal cord injury is promoted in the absence of the axon guidance protein EphA4. Antagonism of EphA4 was therefore proposed as a potential therapy to promote recovery from spinal cord injury. To further assess this potential, two soluble recombinant blockers of EphA4, unclustered ephrin-A5-Fc and EphA4-Fc, were examined for their ability to promote axonal regeneration and to improve functional outcome following spinal cord hemisection in wildtype mice. A 2-week administration of either of these blockers following spinal cord injury was sufficient to promote substantial axonal regeneration and functional recovery by 5 weeks following injury. Both inhibitors produced a moderate reduction in astrocytic gliosis, indicating that much of the effect of the blockers may be due to promotion of axon growth. These studies provide definitive evidence that soluble inhibitors of EphA4 function offer considerable therapeutic potential for the treatment of spinal cord injury and may have broader potential for the treatment of other central nervous system injuries.     

An evaluation of myelomeres and segmentation of the chick embryo spinal cord.

We have investigated whether the neuromeres of the developing chick spinal cord (myelomeres) are manifestations of intrinsic segmentation of the CNS by studying the patterns of cell proliferation and neuronal differentiation. Treatment of 2-day embryos with colchicine does produce exaggerated myelomeres, in confirmation of K?llén (Z. Anat. Entwickl.-Gesch. 123, 309-319, 1962). However, this does not imply that myelomeres are segmental proliferation centres: the undulations caused by colchicine are irregular alongside the unsegmented mesoderm, and another mitotic inhibitor, bromodeoxyuridine, has no such effects. In contrast to lower vertebrate embryos, there is no evidence for segmental groups of primary motor neurons in the chick: the earliest motor neurons express cholinesterase, and project their axons into the adjacent sclerotome, at random positions in relation to the somite boundaries. The population of motor neurons projecting HRP-labelled axons into a single somite lies out of phase with both myelomere and somite, and is placed symmetrically about the anterior half-sclerotome. The earliest intrinsic spinal cord neurons, as stained with zinc iodide-osmium tetroxide or anti-68 x Mr neurofilament antibody, show no segmental patterns of differentiation. We conclude that, in contrast to the rhombomeres of the developing hindbrain, myelomeres are not matched by segmental groupings of differentiating nerve cells, and result from mechanical moulding of the neuroepithelium by the neighbouring somites.     

Retrograde transport of NGF by early chick embryo spinal cord motoneurons

Neuronal retrograde transport of nerve growth factor (NGF) was examined in chick embryos at 5, 6, and 7 days of incubation. Radiolabeled NGF was injected in the target limb muscle and the retrograde transport was viewed following processing for autoradiography. Silver grains were localized in the peripheral nerve, in the ventral root, in neuronal cell bodies within the dorsal root ganglion, and in motoneurons of the lateral motor column. Comparable injections of 125I-cytochrome c resulted in the presence of label at the peripheral injection site only. The possible developmental significance of these observations is discussed.     

MN-cadherin and its novel variant are transiently expressed in chick embryo spinal cord

To isolate cDNAs that are involved in limb-motoneuron development, we compared mRNAs of lumbar and thoracic motoneurons purified from spinal cord of E4 chick embryo by differential display. In situ hybridization demonstrated that one of cDNAs is expressed exclusively in lateral motor column in spinal cord from E4 to E10. We identified two mRNA variants for the cDNA by library screening. The long form (788 amino acids) was identical to chick MN-cadherin. The short variant (543 amino acids) lacks the first two of five extracellular domains of MN-cadherin, which commonly exist in classical cadherins. The amino acid sequence of the short form is identical to that of the carboxyl terminal MN-cadherin, except for the distinct signal sequence. The ratio of mRNA of short form to long form was 1-20. cDNA transfection study revealed that the long form but not the short form MN-cadherin had cell adhesion activity.     

EMG activity of slow and fast ankle extensors following spinal cord transection

Transformations of slow-twitch fibers to the fast-twitch type following spinal cord transection are thought to be related to a substantial decrease or virtual absence of neuromuscular activity. In this experiment, spontaneous activity levels in spinalized and normal cats, raised under similar conditions, were assessed by integrated electromyography (I-EMG) recorded for 240 min over 24 h from the slow-contracting soleus (SOL) and the fast-contracting lateral gastrocnemius (LG). In the SOL of the spinalized cats, there was a 75% reduction in total I-EMG and a 66% reduction in the total duration of muscle activity. Conversely, the LG showed no significant change in total I-EMG, but there was a 66% reduction in the total duration of muscle activity. Based on muscle property data published in companion studies, there was no significant correlation between the SOL total I-EMG and the reduction in contraction times or the decrease in the percentage of slow-twitch fibers determined histochemically. We conclude that transformations of slow-twitch fibers following spinal transection may be regulated by several factors, among which is the total level of spontaneous daily activity.     

Developmental decrease in neurite extension in cultured chick embryo retina and spinal cord neurons

Retina and spinal cord neurons from chick embryos attach to culture substrates and extend neurites. There is a statistically significant age-related decrease in the percentage and average length of neurites formed in 24-hr cultures of chick retina and spinal cord neurons between 6 and 16 days of embryonic age. The developmental decrease of neurite extension may be important for synaptogenesis in the developing nervous system.     

Kinetic characteristics of inhibitory postsynaptic currents in cultured chick embryo spinal cord neurons

Decay of inhibitory postsynaptic currents (IPSC) was analyzed in dissociated culture of chick embryo spinal cord. Differences in the kinetic characteristics of low-amplitude and giant IPSC were revealed. Decay of currents in the first group was single-exponential, while decay in the second group was double-exponential. The time constant of single-exponential current decay increased during membrane depolarization and decreased during rise in temperature of the solution. Decay of the double-exponential currents depended little on potential, while temperature changes acted only on its slow component. Strychnine in submaximum concentrations produced not only a decrease in amplitude of giant IPSC, but also a deceleration of decay due to the slow component. The regularity of these phenomena suggests that decay of giant IPSC, as distinguished from that of low-amplitude currents, is determined by removal of transmitter from the synaptic cleft.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the USSR, Kiev. Translated from Neirofiziologiya, Vol. 23, No. 4, pp. 427–435, July–August, 1991.     

Segmental lineage restrictions in the chick embryo spinal cord depend on the adjacent somites.

We have investigated whether the developing spinal cord is intrinsically segmented in its rostrocaudal (anteroposterior) axis by mapping the spread of clones derived from single labelled cells within the neural tube of the chick embryo. A single cell in the ventrolateral neural tube of the trunk was marked in situ with the fluorescent tracer lysinated rhodamine dextran (LRD) and its descendants located after two days of further incubation. We find that clones derived from cells labelled before overt segmentation of the adjacent mesoderm do not respect any boundaries within the neural tube. Those derived from cells marked after mesodermal segmentation, however, never cross an invisible boundary aligned with the middle of each somite, and tend to be elongated along the mediolateral axis of the neural tube. When the somite pattern is surgically disturbed, neighbouring clones derived from neuroectodermal cells labelled after somite formation behave like clones derived from younger cells: they no longer respect any boundaries, and are not elongated mediolaterally. These results indicate that periodic lineage restrictions do exist in the developing spinal cord of the chick embryo, but their maintenance requires the presence of the adjacent somite mesoderm.     

Stage-dependent restoration of sensory dorsal columns following spinal cord transection in anuran tadpoles

In frogs sensory axons from the lumbar dorsal roots ascend in the dorsal column of the spinal cord to terminate in the medulla and cerebellum. The response of these axons to complete transection of the thoracic spinal cord has been analysed in Rana temporaria tadpoles at different stages of development. The presence and position of dorsal column axons were assessed by using the anterograde transport of horseradish peroxidase or by electrophysiological methods. Before developmental stage VIII, dorsal column axons can grow across the transection and reach their normal areas of termination in the brainstem. Axons that do cross the transection follow their normal pathways. From stage VIII onwards this capacity for growth is largely lost. These results are discussed in terms of the relation between neurogenesis, axon growth and axonal regeneration.     

Effects of combining methylprednisolone with rolipram on functional recovery in adult rats following spinal cord injury

Methylprednisolone (MP) has been widely used as a standard therapeutic agent for the treatment of spinal cord injury (SCI). Because of its controversial beneficial effects, the combination of MP and other pharmacological agents aimed at enhancing functional recovery is desirable. The phosphodiesterase 4 (PDE4) inhibitor rolipram has been implicated in promotion of regeneration due to elevating cAMP. In the present study, we sought to determine the effects of MP and rolipram, administered in combination, after spinal cord injury (SCI) in adult rats. Here we show that in vitro administration of rolipram and MP significantly increased neuron survival and promoted neurite outgrowth of neurons on the inhibitory substrate CSPGs by upregulation of MMP-2 expression; in vivo administration of rolipram and MP inhibited CSPG expression and increase CSPG digestion after rat SCI. Rolipram and MP combining treatment promoted significant neuroprotection through reduced motoneuron death, minimized lesion cavity, and increased regeneration of lesioned corticospinal tract (CST) axons beyond the lesion site after SCI. Enhanced functional recovery was also observed. Overall, our study strongly suggested that the combination treatment of MP and rolipram may represent a promising strategy for clinically applicable pharmacological therapy for rapid initiation of neuroprotection after SCI.