Corticospinal Tract Regeneration Research Articles

Immunization with recombinant Nogo-66 receptor (NgR) promotes axonal regeneration and recovery of function after spinal cord injury in rats

Nogo-66 receptor (NgR), a common receptor for the three known myelin-associated inhibitors, i.e., Nogo-A, myelin-associated glycoprotein (MAG), and oligodendrocyte myelin glycoprotein (OMgp), plays a key role in the failure of axonal regeneration in the adult mammalian central nervous system (CNS). Here we report a novel vaccine approach that stimulates the production of anti-NgR antibody to overcome NgR-mediated growth inhibition after spinal cord injury (SCI). We showed that adult rats immunized with recombinant NgR produced high titers of the anti-NgR antibody and that antisera obtained from the immunized rats promoted neurite outgrowth of rat cerebellar neurons on the inhibitory MAG substrate in vitro. In a spinal cord dorsal hemisection model, NgR immunization promoted regeneration of lesioned corticospinal tract (CST) axons, anterogradely labeled with biotin dextran amine (BDA), beyond the lesion site. In a contusive SCI model, NgR immunization markedly reduced the total lesion volume and improved Basso, Beattie, and Bresnahan (BBB) locomotor rating scale and grid walking performance. Thus, the NgR vaccine approach may represent a promising repair strategy to promote structural and functional recovery following SCI.

Graft of pre-injured sural nerve promotes regeneration of corticospinal tract and functional recovery in rats with chronic spinal cord injury

A possible treatment approach for chronic spinal cord injuries has been tested. We report that minced, autologous, pre-injured peripheral nerve administered as a single injection into an injury-induced cyst, resulting from a contusion injury of the thoracic spinal cord, stimulates recovery of hindlimb locomotor function in rats, as measured by the Basso, Beattie, Bresnahan Locomotor Rating Scale. This response was further enhanced by the addition of exogenous neurotrophic factors. Histological analysis showed axons of the corticospinal tract exhibited significant regeneration past the injury site, when quantified both by number and length. Results indicate that the use of a pre-injured peripheral nerve graft stimulates chronically injured descending nerves to overcome a local inhibitory environment. The resulting sprouting and growth past the injury site is associated with a significant improvement in locomotor function.

Strategies for axonal repair in central nervous system diseases

Significant functional recovery after spinal cord injury can be observed in rodents, in spite of the presence of extensive irreversible structural damage. One potential mechanism for functional regeneration of the corticospinal tract may involve the formation of new spinal circuits remote from the lesion that allow the lesion site to be by-passed and contact re-established with the original targets of the transected axons. In both traumatic and inflammatory lesions of the spinal cord, transection at the thoracic level is followed by extensive collateral sprouting at higher levels of the spinal cord. These collaterals can make contact with long propriospinal neurones in the cervical cord, which project to the lumbar ventral horn via the ventral funiculus. These new connections can serve as a ‘bridge’ to reconnect the corticospinal neurone to its target anterior horn cell. The integrity of this circuitry has been demonstrated by retrograde tracing with pseudorabies virus. Recuperation of electromyographic activity following stimulation of the motor cortex indicates that these new spinal circuits are functionally active. Such remodelling of spinal circuitry following spinal injury may play an important role in determining the extent of functional recovery.

The Nogo–Nogo Receptor Pathway Limits a Spectrum of Adult CNS Axonal Growth

The hypothesis that Nogo-A (Reticulon 4A) and Nogo-66 receptor (NgR1) limit adult CNS axonal growth after injury is supported by both in vitro experiments and in vivo pharmacological studies. However, genetic assessment of the role of Nogo-A in corticospinal tract (CST) axons after spinal cord dorsal hemisection has yielded conflicting results. CST regeneration is detected in homozygous nogo-ab(trap/trap) mice, but not in nogo-ab(atg/atg) mice. CST regeneration is also present after pharmacological NgR blockade, but not in ngr1(-/-) mice. To assess the nogo-ab(atg) and ngr1-null alleles for other axon growth phenotypes, we created unilateral pyramidotomies and monitored the uninjured CST. There is robust pyramidotomy-induced growth of nogo-ab(atg/atg) and ngr1(-/-) CST axons into denervated cervical gray matter. This fiber growth correlates with recovery of fine motor skill in the affected forelimb. Thus nogo-ab and ngr1 play a modulated role in limiting CNS axonal growth across a spectrum of different tracts in various lesion models.

Corticospinal Tract Transection Permanently Abolishes H-Reflex Down-Conditioning in Rats

Previous studies have shown that corticospinal tract (CST) transection, but not transection of other major spinal cord tracts, prevents down-conditioning of the H-reflex, the electrical analog of the spinal stretch reflex. This study set out to determine whether the loss of the capacity for H-reflex down-conditioning caused by CST transection is permanent. Female Sprague-Dawley rats received CST, lateral column (LC), or dorsal column ascending tract (DA) transection at T8-9; 9-10 months later, they were exposed to the H-reflex down-conditioning protocol for 50 days. In the LC and DA rats, H-reflex size fell to 60 (+/- 9 SEM)% and 60 (+/- 19)%, respectively, of its initial size. This down-conditioning was comparable to that of normal rats. In contrast, H-reflex size in the CST rats rose to 170 (+/- 42)% of its initial size. A similar rise does not occur in rats exposed to down-conditioning shortly after CST transection. These results indicate that CST transection permanently eliminates the capacity for H-reflex down-conditioning and has gradual long-term effects on sensorimotor cortex function. They imply that H-reflex down-conditioning can be a reliable measure of CST function for long-term studies of the effects of spinal cord injury and/or for evaluations of the efficacy of experimental therapeutic procedures, such as those intended to promote CST regeneration. The results also suggest that the role of sensorimotor cortex in down-conditioning extends beyond generation of the essential CST activity.

Targeting Neurite Growth Inhibitors to Induce CNS Regeneration

Prominent among the several endogenous inhibitors known to limit recovery and plasticity after CNS injury are Nogo (neurite outgrowth inhibitor) and MAG (myelin associated glycoprotein). The effects of these inhibitors on axonal regeneration can be reduced by administration of specific antagonists, some of which are commercially available for experimental investigation. There are three aspects of therapeutic manipulations: targeting the inhibitory proteins, antagonizing the known receptor, and inhibiting the intracellular signal transduction of these inhibitory molecules. Infusion of an antibody against Nogo improves behavioral deficits and enhances corticospinal tract regeneration in animals after stroke and spinal cord injury (SCI). Similarly, peripheral injection of a mouse monoclonal antibody directed against MAG results in dramatic preferential motor reinnervation in mice after transection of the femoral nerve, indicating that interference with the repellant function of MAG facilitates reinnervation of correct pathways by motor neurons. Further, antagonism of the Nogo receptor by the peptide NEP 1-40 (Nogo extracellular peptide residues 1-40) can promote axonal regeneration in rats after SCI. Blockade of signal transduction also can be effective. The p75 neurotrophin receptor probably represents the signaling part of the receptor complex for neurite growth inhibitors. There is evidence in vitro that the inhibitory actions of MAG and myelin are blocked if neurons are primed with a variety of neurotrophins. Thus, there are several therapeutic approaches to overcome the actions of endogenous neurite growth inhibitors so as to promote CNS regeneration.

Genetic deletion of the Nogo receptor does not reduce neurite inhibition in vitro or promote corticospinal tract regeneration in vivo

Axon regeneration failure in the adult mammalian CNS is attributed in part to the inhibitory nature of CNS myelin. Three myelin-associated, structurally distinct proteins, Nogo, myelin-associated glycoprotein, and oligodendrocyte myelin glycoprotein, have been implicated in this inhibition. Neuronal Nogo receptor (NgR) binds to each of the three inhibitors and has been proposed to mediate their inhibitory signals by complexing with a signal-transducing coreceptor, the neurotrophin receptor p75(NTR). To assess the contribution of NgR to mediating myelin inhibitory signals and regeneration failure in vivo, we generated and characterized NgR-deficient mice. Nogo transcripts are up-regulated in NgR mutants, indicating that NgR regulates Nogo in vivo. However, neurite outgrowth from NgR-deficient postnatal dorsal root ganglion or cerebellar granule neurons is inhibited by myelin and by a Nogo-66 substrate to the same extent as is from wild-type neurons, whereas p75(NTR)-deficient neurons are less inhibited. The NgR ligand-binding domain promotes neurite outgrowth on Nogo-66, regardless of the genotype of the neurons, indicating that the NgR ligand-binding domain can act independent of NgR. Thus, NgR is not essential for mediating inhibitory signals from CNS myelin, at least in the neurons tested, whereas p75(NTR) plays a central role in this response. Neither NgR-nor p75(NTR)-deficient mice showed enhanced regeneration of corticospinal tract axons in comparison with wild-type controls after spinal dorsal hemisection. Our results thus fail to support a central role for NgR in axonal growth inhibition in vitro or in corticospinal tract regeneration block in vivo.

Nogo-66 receptor antagonist peptide promotes axonal regeneration.

Myelin-derived axon outgrowth inhibitors, such as Nogo, may account for the lack of axonal regeneration in the central nervous system (CNS) after trauma in adult mammals. A 66-residue domain of Nogo (Nogo-66) is expressed on the surface of oligodendrocytes and can inhibit axonal outgrowth through an axonal Nogo-66 receptor (NgR). The IN-1 monoclonal antibody recognizes Nogo-A and promotes corticospinal tract regeneration and locomotor recovery; however, the undefined nature of the IN-1 epitope in Nogo, the limited specificity of IN-1 for Nogo, and nonspecific anti-myelin effects have prevented a firm conclusion about the role of Nogo-66 or NgR. Here, we identify competitive antagonists of NgR derived from amino-terminal peptide fragments of Nogo-66. The Nogo-66(1 40) antagonist peptide (NEP1 40) blocks Nogo-66 or CNS myelin inhibition of axonal outgrowth in vitro, demonstrating that NgR mediates a significant portion of axonal outgrowth inhibition by myelin. Intrathecal administration of NEP1 40 to rats with mid-thoracic spinal cord hemisection results in significant axon growth of the corticospinal tract, and improves functional recovery. Thus, Nogo-66 and NgR have central roles in limiting axonal regeneration after CNS injury, and NEP1-40 provides a potential therapeutic agent.

Stimulation of corticospinal tract regeneration in the chronically injured spinal cord.

Acute spinal cord injury models have proved popular in studies aimed at identifying factors capable of influencing axonal regeneration within the central nervous system. In these models, the test factors (e.g. graft tissues or cells, antibodies, growth factors, etc.) are typically administered at the time of spinal cord injury. In this study, we use a rat chronic spinal cord injury model to identify possible factors which can stimulate regeneration of the chronically lesioned corticospinal tract axons. We demonstrate that surgical grafting of segments of autologous, preligated sural nerve, into the syrinx, stimulates sprouting and regeneration of the corticospinal tract as evidenced by the presence of anterograde labelled corticospinal tract processes within the cavity walls two or more weeks after treatment. Regrowing corticospinal processes were not observed within control animals. The anterogradely labelled corticospinal tract axons were found exclusively within the central grey tissue comprising the cavity walls with no regrowing corticospinal process observed within the white matter. A similar pattern of regeneration was observed following injection into the cavity of a suspension of minced autologous preligated sural nerve. Evidence of corticospinal tract regeneration was seen when either wheat germ agglutinin--horseradish peroxidase or biotinylated--dextran was used as an anterograde tracer. These data demonstrate that the chronically injured cortical motor neurons retain the capacity to regenerate for extended periods and that regeneration can be stimulated using grafts of minced, preligated autologous peripheral nerve tissue.

Elimination of Basal Lamina and the Collagen “Scar” after Spinal Cord Injury Fails to Augment Corticospinal Tract Regeneration

The production of specific extracellular matrix molecules is upregulated following injury to the adult CNS, and some of these molecules have been postulated to inhibit axonal regeneration. In particular, the deposition of collagen in conjunction with basal lamina formation has been correlated with the failure of CNS axons to extend beyond sites of injury. In the present experiment, the spatial and temporal distribution of fibrillar collagen type III and the main constituents of basal lamina (collagen type IV and laminin) were characterized after defined lesions of the adult spinal cord at cervical and thoracic levels. The deposition of collagen was then blocked in animals undergoing defined mid-thoracic spinal cord lesions by administration of the iron chelator 2,2′-bipyridine, and subsequent effects on corticospinal axonal growth were examined. At time points from 1 to 6 weeks postinjury, collagen and laminin were deposited at spinal cord lesion sites as a dense matrix at the host–lesion interface that extended for short distances into the surrounding spinal cord parenchyma. The failure of corticospinal axons to grow beyond the lesioned region correlated spatially and temporally with collagen III formation and basal lamina production. However, successful blockade of collagen and basal lamina formation with 2,2′-bipyridine injections failed to enhance corticospinal axon regeneration or sprouting. These results suggest either that collagen and basal lamina formation after CNS injury do not contribute to corticospinal axonal growth failure or, more likely, that molecules in addition to collagen and basal lamina contribute to axonal growth failure and must be collectively blocked to promote corticospinal regeneration.

Collagen implants and cortico-spinal axonal growth after mid-thoracic spinal cord lesion in the adult rat.

We describe an experimental model to study regeneration of lesioned corticospinal tract (CST) fibers in the adult rat spinal cord. After transection of all CST fibers at mid-thoracic level the gap is grafted with a sterile, cell-free collagen matrix. Two methods of collagen-application are used: 1) injection of a fluid collagen solution into the lesioned area which self-assembles in situ and 2) implantation of a solid collagen gel. At 4 weeks post-implantation CST axons are anterogradely labelled with horseradish-peroxidase (HRP). The collagen implant is evaluated for ingrowth of CST axons. The histopathological reaction (gliotic response) around the lesion and within the matrix is also studied. After application of a fluid collagen solution into the lesion area HRP-labelled CST axons can be visualized within the implant. In addition, astroglial and reactive microglial cells invade the collagen-matrix. On the other hand, if collagen is implanted as an already self-assembled gel, no ingrowth of labelled CST axons nor of astroglial/reactive microglial cells is observed. Both methods of collagen-application result in a considerable reduction of the gliotic response as compared to the ungrafted animals. We conclude that the method of application of collagen (i.e., fluid or gel) considerably affects the response of lesioned CST axons. The application of a fluid collagen graft which in situ self-assembles is beneficial for the regrowth of lesioned CST axons in rat spinal cord. In this respect the formation of an astroglial scaffolding structure within the (fluid) collagen, probably due to optimal integration between host and graft, is very important. The inability of injured CST fibers to enter the solid collagen graft may be related to the absence of an astroglial scaffolding structure within the implant.

Axonal regeneration in the rat spinal cord produced by an antibody against myelin-associated neurite growth inhibitors

After lesions in the differentiated central nervous system (CNS) of higher vertebrates, interrupted fibre tracts do not regrow and elongate by more than an initial sprout of approximately 1 mm. Transplantations of pieces of peripheral nerves into various parts of the CNS demonstrate the widespread capability of CNS neurons to regenerate lesioned axons over long distances in a peripheral nerve environment. CNS white matter, cultured oligodendrocytes (the myelin-producing cells of the CNS), and CNS myelin itself, are strong inhibitors of neuron growth in culture, a property associated with defined myelin membrane proteins of relative molecular mass (Mr) 35,000 (NI-35) and 250,000 (NI-250). We have now intracerebrally applied the monoclonal antibody IN-1, which neutralizes the inhibitory effect of both these proteins, to young rats by implanting antibody-producing tumours. In 2-6-week-old rats we made complete transections of the cortico-spinal tract, a major fibre tract of the spinal cord, the axons of which originate in the motor and sensory neocortex. Previous studies have shown a complete absence of cortico-spinal tract regeneration after the first postnatal week in rats, and in adult hamsters and cats. In IN-1-treated rats, massive sprouting occurred at the lesion site, and fine axons and fascicles could be observed up to 7-11 mm caudal to the lesion within 2-3 weeks. In control rats, a similar sprouting reaction occurred, but the maximal distance of elongation rarely exceeded 1 mm. These results demonstrate the capacity for CNS axons to regenerate and elongate within differentiated CNS tissue after the neutralization of myelin-associated neurite growth inhibitors.

Retrograde transport in corticospinal neurons after spinal cord transection.

Complete spinal cord transection at T-6/T-7 in rats caused a decrease in the number of surviving corticospinal neurons. Cell death began 5 and 10 weeks after cord injury. The number of surviving cells decreased progressively for at least 25 weeks after injury. Surviving cells were identified by their ability to transport horseradish peroxidase (HRP) retrograde from a T-1/T-2 insertion site to cortical cell somas. Therapy aimed at promoting corticospinal tract regeneration must be started early after spinal cord injury.

Regeneration of corticospinal axons in the rat

AbstractIn the rat, a few long descending motor tracts capable of carrying an impulse and causing a propagated impulse in the ipsilateral sciatic nerve will regenerate after complete spinal cord transection. In this experiment such regeneration was found in both treated and control animals.Orthograde axonal transport of tritiated proline injected into the motor cortex labels only the corticospinal tracts in the rat spinal cord. Scintillation counts of measured lengths of spinal cord can be used as a measure of the number of labeled corticospinal axons.Comparison of radioactivity per unit length of measured cord segments taken from above and below the site of a previous spinal cord transection can give a reliable estimate of the number of labeled axons that regenerated and crossed the site of injury. Using this test we have demonstrated that some corticospinal axons had regenerated six months after spinlal cord transection in control animals, animals made tolerant to degenerating spinal cord antigens, and animals treated with cyclophosphamide. A group treated with a single 75 mg per kilogram dose of cyclophosphamide 24 hours after spinal cord transection showed the best evidence of corticospinal tract regeneration.