Loren W. Oschipok

Survival and regeneration of rubrospinal neurons 1 year after spinal cord injury.

Scientific interest to find a treatment for spinal cord injuries has led to the development of numerous experimental strategies to promote axonal regeneration across the spinal cord injury site. Although these strategies have been developed in acute injury paradigms and hold promise for individuals with spinal cord injuries in the future, little is known about their applicability for the vast majority of paralyzed individuals whose injury occurred long ago and who are considered to have a chronic injury. Some studies have shown that the effectiveness of these approaches diminishes dramatically within weeks after injury. Here we investigated the regenerative capacity of rat rubrospinal neurons whose axons were cut in the cervical spinal cord 1 year before. Contrary to earlier reports, we found that rubrospinal neurons do not die after axotomy but, rather, they undergo massive atrophy that can be reversed by applying brain-derived neurotrophic factor to the cell bodies in the midbrain. This administration of neurotrophic factor to the cell body resulted in increased expression of growth-associated protein-43 and Tα1 tubulin, genes thought to be related to axonal regeneration. This treatment promoted the regeneration of these chronically injured rubrospinal axons into peripheral nerve transplants engrafted at the spinal cord injury site. This outcome is a demonstration of the regenerative capacity of spinal cord projection neurons a full year after axotomy.

Dose-dependent beneficial and detrimental effects of ROCK inhibitor Y27632 on axonal sprouting and functional recovery after rat spinal cord injury.

Axonal regeneration within the injured central nervous system (CNS) is hampered by multiple inhibitory molecules in the glial scar and the surrounding disrupted myelin. Many of these inhibitors stimulate, either directly or indirectly, the Rho intracellular signaling pathway, providing a strong rationale to target it following spinal cord injuries. In this study, we infused either control (PBS) or a ROCK inhibitor, Y27632 (2 mM or 20 mM, 12 microl/day for 14 days) into the intrathecal space of adult rats starting immediately after a cervical 4/5 dorsal column transection. Histological analysis revealed that high dose-treated animals displayed significantly more axon sprouts in the grey matter distal to injury compared to low dose-treated rats. Only the high dose regimen stimulated sprouting of the dorsal ascending axons along the walls of the lesion cavity. Footprint analysis revealed that the increased base of support normalized significantly faster in control and high dose-treated animals compared to low dose animals. Forepaw rotation angle, and the number of footslips on a horizontal ladder improved significantly more by 6 weeks in high dose animals compared to the other two groups. In a food pellet reaching test, high dose animals performed significantly better than low dose animals, which failed to recover. There was no evidence of mechanical allodynia in any treatment group; however, the slightly shortened heat withdrawal times normalized only with the high dose treatment. Collectively, our data support beneficial effects of high dose Y27632 treatment but indicate that low doses might be detrimental.

Brain-derived neurotrophic factor gene transfer with adeno-associated viral and lentiviral vectors prevents rubrospinal neuronal atrophy and stimulates regeneration-associated gene expression after acute cervical spinal cord injury.

Study Design. Experimental animal study. Objective. To determine if viral vectors carrying the gene for brain-derived neurotrophic factor (BDNF) could be used to promote an axonal regenerative response in rubrospinal neurons after an acute cervical spinal cord injury. Summary of Background Data. Following axotomy in the cervical spinal cord, rubrospinal neurons undergo severe atrophy and fail to up-regulate important genes for regeneration. This can be attenuated or reversed with the infusion of BDNF to the injured cell bodies. This infusion technique, however, causes substantial parenchymal damage around the red nucleus and is limited by occlusion of the infusion pumps. This study examined whether viral vectors could be used to deliver the BDNF gene in a less damaging fashion and whether this could promote a regenerative response in injured rubrospinal neurons. Methods. Following a cervical spinal cord injury, the viral vectors were injected into the vicinity of the injured red nucleus. The extent of parenchymal damage around the red nucleus was assessed, as was the immunoreactivity to BDNF and cellular transfection patterns. Rubrospinal neuronal cross-ectional area was measured to determine if atrophy had been reversed, and in situ hybridization for GAP-43 and T&agr;1 tubulin was performed to determine if there genes, which are important for axonal regeneration, were up-egulated. Results. Parenchymal damage associated with viral injection was significantly less than with previous infusion techniques. BDNF immunoreactivity around the red nucleus indicated that the BDNF transgene was expressed. Both viral vectors reversed rubrospinal neuronal atrophy and promoted the expression of GAP-43 and T&agr;1 tubulin. Conclusions. Viral-ediated transfer of the BDNF gene was successful at promoting a regenerative response in rubrospinal neurons following acute cervical spinal cord injury, with significantly less parenchymal damage than previously observed when infusing the BDNF protein.

Rubrospinal neurons fail to respond to brain-derived neurotrophic factor applied to the spinal cord injury site 2 months after cervical axotomy.

Numerous experimental therapies to promote axonal regeneration have shown promise in animal models of acute spinal cord injury, but their effectiveness is often found to diminish with a delay in administration. We evaluated whether brain-derived neurotrophic factor (BDNF) application to the spinal cord injury site 2 months after cervical axotomy could promote a regenerative response in chronically axotomized rubrospinal neurons. BDNF was applied to the spinal cord in three different concentrations 2 months after cervical axotomy of the rubrospinal tract. The red nucleus was examined for reversal of neuronal atrophy, GAP43 and Talpha1 tubulin mRNA expression, and trkB receptor immunoreactivity. A peripheral nerve transplant paradigm was used to measure axonal regeneration into peripheral nerve transplants. Rubrospinal axons were anterogradely traced and trkB receptor immunohistochemistry performed on the injured spinal cord. We found that BDNF treatment did not reverse rubrospinal neuronal atrophy, nor promote GAP-43 and Talpha1 tubulin mRNA expression, nor promote axonal regeneration into peripheral nerve transplants. TrkB receptor immunohistochemistry demonstrated immunoreactivity on the neuronal cell bodies, but not on anterogradely labeled rubrospinal axons at the injury site. These findings suggest that the poor response of rubrospinal neurons to BDNF applied to the spinal cord injury site 2 months after cervical axotomy is not related to the dose of BDNF administered, but rather to the loss of trkB receptors on the injured axons over time. Such obstacles to axonal regeneration will be important to identify in the development of therapeutic strategies for chronically injured individuals.

Galectin-1 in regenerating motoneurons.

The exogenous application of recombinant galectin‐1 has recently been shown to promote the rate of peripheral nerve regeneration. Endogenous neuronal galectin‐1 expression has recently been demonstrated to increase after axotomy. Here we demonstrate a significant increase in the endogenous neuronal expression of galectin‐1 mRNA in facial motoneurons after either a nerve resection or crush injury in mice. This increase in galectin‐1 expression was due in part to the loss of target‐derived factor(s) as indicated by both the return of galectin‐1 expression to control levels following target re‐innervation and the increase in galectin‐1 expression after blockade of axonal transport by an interneuronal colchicine injection. Furthermore, interneuronal injections of glial‐derived neurotrophic factor into the uninjured nerve also increased galectin‐1 mRNA expression within facial motoneurons suggesting that positive signals may also be involved in the regulation of galectin‐1 expression. Galectin‐1 null mutant mice showed an attenuated rate of functional recovery of whisking movement after a facial nerve crush.

Altered primary afferent anatomy and reduced thermal sensitivity in mice lacking galectin-1

&NA; The transmission of nociceptive information occurs along non‐myelinated, or thinly myelinated, primary afferent axons. These axons are generally classified as peptidergic (CGRP‐expressing) or non‐peptidergic (IB4‐binding), although there is a sub‐population that is both CGRP‐positive and IB4‐binding. During neuronal development and following injury, trophic factors and their respective receptors regulate their survival and repair. Recent reports also show that the carbohydrate‐binding protein galectin‐1 (Gal1), which is expressed by nociceptive primary afferent neurons during development and into adulthood, is involved in axonal pathfinding and regeneration. Here we characterize anatomical differences in dorsal root ganglia (DRG) of Gal1 homozygous null mutant mice (Gal1−/−), as well as behavioural differences in tests of nociception. Gal1−/− mice have a significantly reduced proportion of IB4‐binding DRG neurons, an increased proportion of NF200‐immunoreactive DRG neurons, increased depth of central terminals of IB4‐binding and CGRP‐immunoreactive axons in the dorsal horn, and a reduced number of Fos‐positive second order neurons following thermal (cold or hot) stimulation. While there is no difference in the total number of axons in the dorsal root of Gal1−/− mice, there are an increased number of myelinated axons, suggesting that in the absence of Gal1, neurons that are normally destined to become IB4‐binding instead become NF200‐expressing. In addition, mice lacking Gal1 have a decreased sensitivity to noxious thermal stimuli. We conclude that Gal1 is involved in nociceptive neuronal development and that the lack of this protein results in anatomical and functional deficits in adulthood.

Galectin-1 expression correlates with the regenerative potential of rubrospinal and spinal motoneurons.

Axotomized spinal motoneurons are able to regenerate to their peripheral targets, whereas injured rubrospinal neurons that lie completely within the CNS fail to regenerate. The differing cell body reactions to axotomy of these two neuronal populations have been implicated in their disparate regenerative ability. Recently, the lectin galectin-1 has been shown to be involved in both spinal motoneurons and primary afferent regeneration. Using in situ hybridization, we compared the endogenous galectin-1 mRNA expression in spinal motoneurons and rubrospinal neurons after axotomy. We found that 7 and 14 days after axotomy, galectin-1 mRNA increased in spinal motoneurons but decreased in rubrospinal neurons. Infusion of the brain-derived neurotrophic factor into the vicinity of the injured rubrospinal nucleus, which we have previously shown to increase the regenerative capacity of rubrospinal neurons, significantly increased galectin-1 mRNA compared with uninjured control levels. Thus, the expression of galectin-1 in neurons correlates with the regenerative propensity.

Regulation of neuronal and glial galectin-1 expression by peripheral and central axotomy of rat primary afferent neurons

Galectin-1 (Gal1) is an endogenously-expressed protein important for the embryonic development of the full complement of primary sensory neurons and their synaptic connections in the spinal cord. Gal1 also promotes axonal regeneration following peripheral nerve injury, but the regulation of Gal1 by axotomy in primary afferent neurons has not yet been examined. Here, we show by immunohistochemistry and in situ hybridization that Gal1 expression is differentially regulated by peripheral nerve injury and by dorsal rhizotomy. Following peripheral nerve injury, the proportion of Gal1-positive DRG neurons was increased. An increase in the proportion of large-diameter DRG neurons immunopositive for Gal1 was paralleled by an increase in the depth of immunoreactivity in the dorsal horn, where Gal1-positive terminals are normally restricted to laminae I and II. Dorsal rhizotomy did not affect the proportions of neurons containing Gal1 mRNA or protein, but did deplete the ipsilateral dorsal horn of Gal1 immunoreactivity, indicating that it is transported centrally by dorsal root axons. Dorsal rhizotomy also resulted in an increase in Gal1 mRNA the nerve peripheral to the PNS-CNS interface (likely within Schwann cells and/or macrophages), and to a lesser extent within deafferented spinal cord regions undergoing Wallerian degeneration. This latter increase was notable in the dorsal columns and along the prior trajectories of myelinated afferents into the deeper dorsal horn. These results show that neuronal and glial expressions of Gal1 are tightly correlated with regenerative success. Thus, the differential expression pattern of Gal1 following peripheral axotomy and dorsal rhizotomy suggests that endogenous Gal1 may be a factor important to the regenerative response of injured axons.

Reaxotomy of Chronically Injured Rubrospinal Neurons Results in Only Modest Cell Loss

Among the most promising therapeutic strategies to facilitate axonal regeneration after spinal cord injury is the transplantation of various cellular substrates into the injury site. With the establishment of a glial scar and cyst at the injury site over time, the implantation of such cells in the chronic injury setting may require some resection of these nonpermissive elements, which could concomitantly reinjure already severed axons. This study evaluates the response of chronically injured rubrospinal neurons to such a second axotomy. Our findings indicate that the second axotomy does not lead to an accelerated loss of rubrospinal neurons, which represents an important finding for those who evaluate axonal regeneration of this motor system in chronic transplantation studies.

Both positive and negative factors regulate gene expression following chronic facial nerve resection.

Previously, we reported that following a chronic nerve resection, removal of the neuroma reversed the atrophy, increased the number of countable motoneurons and resulted in the re-expression of GAP-43 and alpha tubulin mRNA. In the present study, we questioned whether this response was due to the removal of the neuroma, or a result of factors such as neurotrophins, produced at the injury site. To test this hypothesis, 10 weeks after axotomy, the axonal transport blocker colchicine or, glial derived neurotrophic factor (GDNF) was injected proximal to the neuroma. The injection of GDNF or colchicine elicited an increase in motoneuron size and in GAP-43, but not alpha tubulin, mRNA. These data suggest that in addition to factors produced at the injury site, the neuroma acts as a source of target-like repressive signals that when removed results in an increase in gene expression and motoneuron size. To analyze the regenerative potential of chronically resected motoneurons, mice without a previous nerve injury and mice with a chronic resection received a pre-degenerated segment of sciatic nerve attached to the proximal facial nerve stump. Axons from both the chronic and acute groups grew into the grafts, however, significantly more retrogradely labeled motoneurons were counted in the acute group compared to the chronic resection group. No difference in motoneuron cell size was observed between the two groups of regenerated neurons. Therefore, despite severe atrophy, many of the surviving mouse facial motoneurons retain the propensity to extend their axons when provided with the appropriate environment.