Wnt produced by stretched roof-plate cells is required for the promotion of cell proliferation around the central canal of the spinal cord.

Cell morphology changes dynamically during embryogenesis, and these changes create new interactions with surrounding cells, some of which are presumably mediated by intercellular signaling. However, the effects of morphological changes on intercellular signaling remain to be fully elucidated. In this study, we examined the effect of morphological changes in Wnt-producing cells on intercellular signaling in the spinal cord. After mid-gestation, roof-plate cells stretched along the dorsoventral axis in the mouse spinal cord, resulting in new contact at their tips with the ependymal cells that surround the central canal. Wnt1 and Wnt3a were produced by the stretched roof-plate cells and delivered to the cell process tip. Whereas Wnt signaling was activated in developing ependymal cells, Wnt activation in dorsal ependymal cells, which were close to the stretched roof plate, was significantly suppressed in embryos with roof plate-specific conditional knockout of Wls, which encodes a factor that is essential for Wnt secretion. Furthermore, proliferation of these cells was impaired in Wls conditional knockout mice during development and after induced spinal cord injury in adults. Therefore, morphological changes in Wnt-producing cells appear to generate new Wnt signal targets.

Prenatal repair of myelomeningocele with aligned nanofibrous scaffolds-a pilot study in sheep.

BACKGROUND/PURPOSE: Spinal cord damage in myelomeningocele (MMC) results from abnormal cord development and subsequent local trauma. Prenatal surgery prevents additional neural injury. However, existing damage is not reversed. Biodegradable nanofibrous scaffolds (NSs) promote regeneration of neural tissues. They mimic the microtopography of the extracellular matrix and guide tissue formation and organization. The purpose of this pilot study was to evaluate the practicality and safety of using biodegradable NS as a regenerative device in prenatal MMC repair. METHODS: Two fetal lambs underwent a surgically induced MMC defect followed by open fetal repair using aligned biodegradable NS. Lambs were killed at day 138. Spinal cords were examined for inflammation or fibrosis and stained for spinal cord architecture, myelin, and neuron cell bodies. RESULTS: Prenatal repair with NS demonstrated technical feasibility. There was no evidence of a surrounding inflammatory response or foreign-body reaction to the scaffold. CONCLUSION: Biodegradable NS can be used surgically for the prenatal repair of MMC in a large animal model and does not appear to elicit an inflammatory or fibrotic reaction in fetal tissue. Further studies will determine their potential for neural cell infiltration, delivery of growth factors, drugs or stem cells, and functional recovery greater than standard repair.

Protein deiminases: new players in the developmentally regulated loss of neural regenerative ability.

Spinal cord regenerative ability is lost with development, but the mechanisms underlying this loss are still poorly understood. In chick embryos, effective regeneration does not occur after E13, when spinal cord injury induces extensive apoptotic response and tissue damage. As initial experiments showed that treatment with a calcium chelator after spinal cord injury reduced apoptosis and cavitation, we hypothesized that developmentally regulated mediators of calcium-dependent processes in secondary injury response may contribute to loss of regenerative ability. To this purpose we screened for such changes in chick spinal cords at stages of development permissive (E11) and non-permissive (E15) for regeneration. Among the developmentally regulated calcium-dependent proteins identified was PAD3, a member of the peptidylarginine deiminase (PAD) enzyme family that converts protein arginine residues to citrulline, a process known as deimination or citrullination. This post-translational modification has not been previously associated with response to injury. Following injury, PAD3 up-regulation was greater in spinal cords injured at E15 than at E11. Consistent with these differences in gene expression, deimination was more extensive at the non-regenerating stage, E15, both in the gray and white matter. As deimination paralleled the extent of apoptosis, we investigated the effect of blocking PAD activity on cell death and deiminated-histone 3, one of the PAD targets we identified by mass-spectrometry analysis of spinal cord deiminated proteins. Treatment with the PAD inhibitor, Cl-amidine, reduced the abundance of deiminated-histone 3, consistent with inhibition of PAD activity, and significantly reduced apoptosis and tissue loss following injury at E15. Altogether, our findings identify PADs and deimination as developmentally regulated modulators of secondary injury response, and suggest that PADs might be valuable therapeutic targets for spinal cord injury.

Post-translational regulation of Crmp in developing and regenerating chick spinal cord.

It is becoming apparent that regulation at the protein level plays crucial roles in developmental and pathological processes. Therefore, we performed a proteomics screen to identify proteins that are differently expressed or modified at stages of development permissive (E11) and nonpermissive for regeneration (E15) of the chick spinal cord. Proteins regulated either developmentally or in response to spinal-cord injury included collapsin-response-mediator proteins (Crmps), known to modulate microtubule dynamic and axonal growth. No significant changes in Crmp transcripts following injury were observed, indicating regulation mainly at the protein level. Analysis of Crmp-2 protein and its phosphorylated forms, pS522 and pT514, showed that Crmp-2 is developmentally regulated and also expressed in neural progenitors in vivo and in neurospheres. Its cellular localization changed both with development and following spinal-cord injury. In addition, although overall levels of Crmp-2 expression were not affected by injury, abundance of certain phosphorylated forms was altered. pT514 Crmp-2 appeared to be associated with dividing neural progenitors and was greatly reduced at nonpermissive stages for regeneration, whereas it did not seem affected by injury. In contrast, phosphorylation of Crmp-2 at S522 was upregulated early after injury in regenerating spinal cords and the ratio between phosphorylated to total Crmp-2 increased, as indicated by 2D Western blots. Altogether, this study shows highly dynamic regulation of Crmp-2 forms during development and identifies post-translational changes in Crmp-2 as putative contributors to the maintenance of spinal-cord regenerative ability, possibly via a transient stabilization of the neuronal cytoskeleton.

Modulation of dendritic spine remodeling in the motor cortex following spinal cord injury: effects of environmental enrichment and combinatorial treatment with transplants and neurotrophin-3.

Incomplete spinal cord injury (SCI) elicits structural plasticity of the spared motor system, including the motor cortex, which may underlie some of the spontaneous recovery of motor function seen after injury. Promoting structural plasticity may become an important component of future strategies to improve functional outcomes. We have recently observed dynamic changes in the density and morphology of dendritic spines in the motor cortex following SCI. The present study sought to test whether SCI-induced changes in spine density and morphology could be modulated by potential strategies to enhance functional recovery. We examined the effects of enriched environment, transplants, and neurotrophin-3 on the plasticity of synaptic structures in the motor cortex following SCI. Housing rats in an enriched environment increased spine density in the motor cortex regardless of injury. SCI led to a more slender and elongated spine morphology. Enriched housing mitigated the SCI-induced morphological alterations, suggesting that the environmental modification facilitates maturation of synaptic structures. Transplantation of embryonic spinal cord tissue and delivery of neurotrophin-3 at the injury site further increased spine density when combined with enriched housing. This combinatorial treatment completely abolished the injury-induced changes, restoring a preinjury pattern of spine morphology. These results demonstrated that remodeling of dendritic spines in the motor cortex after SCI can be modulated by enriched housing, and the combinatorial treatment with embryonic transplants and neurotrophin-3 can potentiate the effects of enriched housing. We suggest that synaptic remodeling processes in the motor cortex can be targeted for an intervention to enhance functional recovery after SCI.

Effects of an embryonic repair graft on recovery from spinal cord injury.

It is widely believed that mammalian CNS axons have little regenerative capacity because their environment is non-permissive to regrowth. This viewpoint is based, in large part, on the fact that in virtually all previous studies on regeneration following spinal cord injury, regenerated axonal projections have been few in number, quite short, and considered to be mostly aberrant. As a result, motor recovery has been very limited in both experimental preparations and the human. In this chapter, we describe use of a neonatal, spinally transected animal model in which selected spinal cord segments were carefully replaced with equivalent tissue from embryonic tissue of the same species. We demonstrate that the new spinal environment is indeed permissive, and reconstruction is possible of neural connections, which are similar to the pre-injury, normal projections. Moreover, the distribution and number of regenerated axons are closely related to the extent of functional motor recovery. Our results suggest that contrary to doctrinaire thought, the mammalian CNS possesses a remarkable capacity for regrowth. For this to be efficacious, however, regenerating axons must contact the inherent, pre-injury guidance system, whose cues were used for establishing appropriate neural connections in the developing animal, and are retained in the adult. It is argued that by use of these guidance cues, regenerating axons that traverse the site of a spinal cord injury, can project on to locate their pre-injury pathways and targets, and thereby restore function.

[The reactions of different sections of human embryonic neuroepithelium to damage to the ventricular surface during explantation]. / Reaktsii razlichnykh uchastkov neiroépiteliia émbrionov cheloveka na povrezhdenie ventrikuliarnoi poverkhnosti pri éksplantatsii.

Fragments of different regions of brain, spinal cord and retina obtained from 6 up to 7 wk human embryos were placed into media 199. On 24th hr of cultivation multiple invaginations developed in the explant ventricular surface as a response to its damage while preparing to explantation. Maturity extent of neural and glial elements was not essential to invagination intensity. Fragments of tectum and retina that are characterised by permanent shape of ventricular surface displayed minimal activity. These data suggest that the reaction considered reflects a property of neuroepithelial cells that constitutes an important factor in normal brain morphogenesis.

The fetal spinal cord does not regenerate after in utero transection in a large mammalian model.

OBJECTIVE: Regeneration and functional recovery after spinal cord transection do not occur in mammalian animals and humans postnatally. The goal of this study was to test whether in utero transection of the fetal spinal cord is succeeded by anatomic healing and functional recovery. METHODS: In five sheep fetuses, at 60 days of gestation and 75 days of gestation (term = 150 d), the spinal cord was completely transected at T10. The animals were delivered near term by cesarean section for clinical evaluation, measurement of cortical somatosensory evoked potentials, and morphological assessment. RESULTS: The newborn lambs demonstrated sensory-motor paraplegia, were incontinent of urine and stool, and exhibited a spinally generated, ambulatory pattern of the hindlimbs. No cortical somatosensory evoked potentials could be recorded in response to posterior tibial nerve stimulation, although potentials from the ulnar nerve, which enters the cord rostral to the lesion, were normal in all animals. Histologically, no neuronal connections across the transection site were identified. The cord proximal to the lesion was grossly normal, whereas distal to the transection, it appeared slightly smaller but with the cytoarchitecture preserved. CONCLUSIONS: Unlike in lower vertebrate and avian species, the fetal ovine spinal cord has no detectable spontaneous regenerative capabilities when transected during midgestation. Gap formation after transection, secondary posttraumatic cell death, and missing guiding channels for sprouting axons may be factors involved in the absence of any regenerative response.

Repair of connections in injured neonatal and embryonic spinal cord in vitro.

A remarkable preparation for studying development and repair is the CNS of the newborn opossum which, removed in its entirety, survives in culture for more than 1 week. In suitable medium, cells continue to divide, mature and reflex activity is maintained. Moreover, nerve fibers grow rapidly, reliably and extensively across lesions made in the spinal cord. Restoration of conduction has been demonstrated by recording electrically; labeled fibres have been observed directly by light and electron microscopy as they traverse the lesion. Similar experiments have also been made in embryonic (E15) rat CNS in culture. Open questions concern the identity of the fibers that traverse the lesion and the specificity of connections that they make with targets. We are now also analysing mechanisms that favor repair in younger opossums and that prevent it in their older siblings. Of particular interest are oligodendrocytes and myelin that start to appear at about 8-9 days after birth.

Recovery of function after spinal cord injury: mechanisms underlying transplant-mediated recovery of function differ after spinal cord injury in newborn and adult rats.

Fetal spinal cord transplants placed into the site of spinal cord injury support axonal growth of host systems in both newborn and adult animals. The amount of axonal growth, however, is much more robust in the newborn animals. The current studies were designed to determine if the differences in the magnitude of the anatomical plasticity of host pathways in the presence of transplants is reflected in differences in recovery of function between the neonatal and adult operates. Newborn and adult rats received a midthoracic "overhemisection." Immediately following the hemisection embryonic (E14) spinal cord transplants were placed into the lesion site. All animals were trained and tested as adults, on a battery of qualitative and quantitative tests of motor function. Immunocytochemical methods were used to compare the extent of growth of descending (serotonergic and noradrenergic) and segmental (calcitonin gene-related peptide containing dorsal root axons) pathways in both groups. The growth of descending pathways into the transplants was substantially greater in density and spatial extent after lesions at birth than at maturity. The distribution of segmental dorsal root axons, in contrast, was similar in both groups. Fetal spinal cord transplants promoted recovery of motor function in both newborn and adult operates. The particular aspects of locomotor function which recover differ between the neonatal and adult operates, suggesting that the mechanisms underlying recovery of function must differ between the two groups.

Focal necrosis of the spinal cord in utero.

We examined a child who suffered in utero necrosis of the spinal cord. Necrosis of this type typically occurs in the fetus with neck hyperextension and breech position and presumably results from focal ischemia of the spinal cord.

Fetal spinal-cord injury secondary to hyperextension of the neck: no effect of caesarean section.

A 24-month-old Japanese girl is reported who had upper spinal-cord injury secondary to fetal hyperextension of the neck in breech presentation. She was first noted to be in this position 10 days before the expected date of birth and was delivered by caesarean section.