Myelin inhibitors, including MAG, are major impediments to CNS regeneration. However, CNS axons of DRGs regenerate if the peripheral branch of these neurons is lesioned first. We show that 1 day post-peripheral-lesion, DRG-cAMP levels triple and MAG/myelin no longer inhibit growth, an effect that is PKA dependent. By 1 week post-lesion, DRG-cAMP returns to control, but growth on MAG/myelin improves and is now PKA independent. Inhibiting PKA in vivo blocks the post-lesion growth on MAG/myelin at 1 day and attenuates it at 1 week. Alone, injection of db-cAMP into the DRG mimics completely a conditioning lesion as DRGs grow on MAG/myelin, initially, in a PKA-dependent manner that becomes PKA independent. Importantly, DRG injection of db-cAMP results in extensive regeneration of dorsal column axons lesioned 1 week later. These results may be relevant to developing therapies for spinal cord injury.

There is little axonal growth after central nervous system (CNS) injury in adult mammals. The administration of antibodies (IN-1) to neutralize the myelin-associated neurite growth inhibitory proteins leads to long-distance regrowth of a proportion of CNS axons after injury. Our aim was: to determine if spinal cord lesion in adult rats, followed by treatment with antibodies to neurite growth inhibitors, can lead to regeneration and anatomical plasticity of other spinally projecting pathways; to determine if the anatomical projections persist at long survival intervals; and to determine whether this fibre growth is associated with recovery of function. We report here that brain stem-spinal as well as corticospinal axons undergo regeneration and anatomical plasticity after application of IN-1 antibodies. There is a recovery of specific reflex and locomotor functions after spinal cord injury in these adult rats. Removal of the sensorimotor cortex in IN-1-treated rats 2-3 months later abolished the recovered contact-placing responses, suggesting that the recovery was dependent upon the regrowth of these pathways.

Neural progenitor cells, including neural stem cells, are a potential expandable source of graft material for transplantation aimed at repairing the damaged CNS. Here we present the first evidence that in vitro-expanded fetusderived neurosphere cells were able to generate neurons in vivo and improve motor function upon transplantation into an adult rat spinal-cord-contusion injury model. As the source of graft material, we used a neural stem cell-enriched population that was derived from rat embryonic spinal cord (E14.5) and expanded in vitro by neurosphere formation. Nine days after contusion injury, these neurosphere cells were transplanted into adult rat spinal cord at the injury site. Histological analysis 5 weeks after the transplantation showed that mitotic neurogenesis occurred from the transplanted donor progenitor cells within the adult rat spinal cord, a nonneurogenic region; that these donor-derived neurons extended their processes into the host tissues; and that the neurites formed synaptic structures. Furthermore, analysis of motor behavior using a skilled reaching task indicated that the treated rats showed functional recovery. These results indicate that in vitro-expanded neurosphere cells derived from the fetal spinal cord are a potential source for transplantable material for treatment of spinal cord injury.

Unlike neonatal axons, mammalian adult axons do not regenerate after injury. Likewise, myelin, a major factor in preventing regeneration in the adult, inhibits regeneration from older but not younger neurons. Identification of the molecular events responsible for this developmental loss of regenerative capacity is believed key to devising strategies to encourage regeneration in adults after injury. Here, we report that the endogenous levels of the cyclic nucleotide, cAMP, are dramatically higher in young neurons in which axonal growth is promoted both by myelin in general and by a specific myelin component, myelin-associated glycoprotein (MAG), than in the same types of neurons that, when older, are inhibited by myelin-MAG. Inhibiting a downstream effector of cAMP [protein kinase A (PKA)] prevents myelin-MAG promotion from young neurons, and elevating cAMP blocks myelin-MAG inhibition of neurite outgrowth in older neurons. Importantly, developmental plasticity of spinal tract axons in neonatal rat pups in vivo is dramatically reduced by inhibition of PKA. Thus, the switch from promotion to inhibition by myelin-MAG, which marks the developmental loss of regenerative capacity, is mediated by a developmentally regulated decrease in endogenous neuronal cAMP levels.

The present study provides the first evidence that signaling occurs between B-ephrins and EphB receptors in the adult CNS in response to injury. Specifically, our combined histological and biochemical data indicate that two members of the B-class of ephrins and Eph receptors, ephrin-B2 and EphB2, are expressed by astrocytes and meningeal fibroblasts, respectively, in the adult spinal cord. In response to thoracic spinal cord transection lesions, ephrin-B2 and EphB2 protein levels exhibit an initial decrease (1 d after lesion), followed by a significant increase by day 14. Immunohistochemical data indicate that ephrin-B2 is expressed by reactive CNS astrocytes, and EphB2 is present on fibroblasts invading the lesion site from the adjacent meninges. During the first 3 d after injury, there is intermingling of ephrin-B2-expressing reactive astrocytes at the lesion surface with EphB2-containing fibroblasts that is concurrent with bidirectional activation (phosphorylation) of ephrin-B2 and EphB2. By 7 d, both cell types are establishing restricted cellular domains containing dense networks of cells and interweaving processes. This astroglial-meningeal fibroblast scar is fully developed by day 14 when there is strict segregation of ephrin-B2-expressing astrocytes from EphB2-positive meningeal fibroblasts. These morphological changes are concomitant with a simultaneous decrease in ephrin-B2 and EphB2 activation. These observations provide strong evidence that cell contact-mediated bidirectional signaling between ephrin-B2 on reactive astrocytes and EphB2 on meningeal fibroblasts is an early event in the cellular cascades that result in the development of the glial scar and the exclusion of meningeal fibroblasts from the injured spinal cord.