Human adult olfactory neural progenitors rescue axotomized rodent rubrospinal neurons and promote functional recovery

Previously, our lab reported the isolation of patient-specific neurosphere-forming progenitor lines from human adult olfactory epithelium from cadavers as well as patients undergoing nasal sinus surgery. RT-PCR and ELISA demonstrated that the neurosphere-forming cells (NSFCs) produced BDNF. Since rubrospinal tract (RST) neurons have been shown to respond to exogenous BNDF, it was hypothesized that if the NSFCs remained viable following engraftment into traumatized spinal cord, they would rescue axotomized RS neurons from retrograde cell atrophy and promote functional recovery. One week after a partial cervical hemisection, GFP-labeled NSFCs suspended in Matrigel matrix or Matrigel matrix alone was injected into the lesion site. GFP-labeled cells survived up to 12 weeks in the lesion cavity or migrated within the ipsilateral white matter; the apparent number and mean somal area of fluorogold (FG)-labeled axotomized RST neurons were greater in the NSFC-engrafted rats than in lesion controls. Twelve weeks after engraftment, retrograde tracing with FG revealed that some RST neurons regenerated axons 4-5 segments caudal to the engraftment site; anterograde tracing with biotinylated dextran amine confirmed regeneration of RST axons through the transplants within the white matter for 3-6 segments caudal to the grafts. A few RST axons terminated in gray matter close to motoneurons. Matrix alone did not elicit regeneration. Behavioral analysis revealed that NSFC-engrafted rats displayed better performance during spontaneous vertical exploration and horizontal rope walking than lesion Matrigel only controls 11 weeks post transplantation. These results emphasize the unique potential of human olfactory neuroepithelial-derived progenitors as an autologous source of stem cells for spinal cord repair.     

Transplants of fibroblasts genetically modified to express BDNF promote axonal regeneration from supraspinal neurons following chronic spinal cord injury

Transplants of fibroblasts genetically modified to express BDNF (Fb/BDNF) have been shown to promote regeneration of rubrospinal axons and recovery of forelimb function when placed acutely into the injured cervical spinal cord of adult rats. Here we investigated whether Fb/BDNF cells could stimulate supraspinal axon regeneration and recovery after chronic (4 week) injury. Adult female Sprague-Dawley rats received a complete unilateral hemisection injury at the third cervical spinal cord segment (C3). Four-five weeks later the injury site was exposed and rats received transplants of unmodified fibroblasts (Fb/UM) or Fb/BDNF. Four-five weeks after transplantation, locomotor recovery was examined on a test of forelimb usage and regeneration of supraspinal axons was studied following injection of the anterograde tracer biotin dextran amine (BDA). Rubrospinal tract (RST), reticulospinal tract (ReST), and vestibulospinal tract (VST) axons regenerated into transplants of either Fb/UM or Fb/BDNF but the length of axonal growth was significantly different in the two groups. The absolute distance of ReST growth was 1.8-fold greater in Fb/BDNF than in Fb/UM and the absolute distance of growth of RST and VST axons showed a statistically significant 4-fold increase. All three types of regenerated axons occupied a greater proportional length of Fb/BDNF transplants than of Fb/UM transplants. Only VST axons extended into the host spinal cord caudal to the Fb/BDNF grafts, but these axons were sparse. Rats receiving Fb/BDNF used both forelimbs together to explore walls of a cylinder more often than rats receiving Fb/UM, indicating partial recovery of forelimb usage. These results demonstrate that fibroblasts genetically modified to express BDNF promote axon regeneration from supraspinal neurons in the chronically injured spinal cord with accompanying partial recovery of locomotor performance.     

Grafts of BDNF-producing fibroblasts rescue axotomized rubrospinal neurons and prevent their atrophy

We have reported that intraspinal transplants of fibroblasts genetically modified to express brain-derived neurotrophic factor (BDNF) promote rubrospinal axon regeneration and functional recovery following subtotal cervical hemisection that completely ablated the rubrospinal tract. In the present study we examined whether these transplants could prevent cell loss and/or atrophy of axotomized Red nucleus neurons. Adult rats received a subtotal spinal cord cervical hemisection followed by a graft of unmodified fibroblasts or fibroblasts producing BDNF into the lesion cavity. One or 2 months later, fluorogold was injected several segments caudal to the lesion-transplant site to retrogradely label those Red nucleus neurons whose axons have regenerated. Unmodified fibroblasts failed to protect against either cell loss or atrophy. Neuron counts and soma-size measurements in Nissl-stained preparations showed a 45% loss of recognizable neurons and 40% atrophy of the surviving neurons in the injured Red nucleus. Grafts of BDNF-producing fibroblasts reduced neuron loss to less than 15% and surviving neurons showed only a 20% decrease in mean soma size. Soma size analysis of fluorogold-labeled Red nucleus neurons indicated that the Red nucleus neurons whose axons regenerated caudal to the graft did not atrophy. We conclude that fibroblasts engineered ex vivo to secrete BDNF and grafted into a partial cervical hemisection promote axon regeneration while reducing cell loss and atrophy of neurons in the Red nucleus. These results suggest that transplants of genetically engineered cells could be an important tool for delivery of therapeutic factors that contribute to the repair of spinal cord injury.     

Fibroblasts genetically modified to produce BDNF support regrowth of chronically injured serotonergic axons

Cells genetically modified to release a variety of growth and/or neurotrophic factors have been used for transplantation into the injured spinal cord as a means to deliver therapeutic products. Axon growth into and through such transplants has been demonstrated after intervention after an acute injury. The present study examined their potential to support regeneration in a chronic injury condition. Five weeks after a cervical hemisection in adult rats, the lesion site was debrided of scar tissue and expanded in both rostral and caudal directions. Animals received a transplant of cultured normal fibroblasts (control) or fibroblasts genetically modified to produce brain-derived neurotrophic factor (BDNF). Six weeks later, animals were killed to determine the extent of growth of serotonergic axons into the transplant. Axons immunoreactive for serotonin (5-HT-ir) were found to cross the rostral interface of host spinal cord readily with either type of fibroblast cell transplant, but the number and density of 5-HT-ir axons extending into the BDNF-producing transplants was markedly greater than those in the control fibroblasts. Axons coursed in all directions among normal fibroblast transplants, whereas growth was more oriented along a longitudinal plane when BDNF was being released by the transplanted cells. The length of growth and the percentage of the transplant length occupied by 5-HT-ir axons were significantly greater in BDNF-producing transplants than in the normal fibroblasts. Many serotonergic axons approached the caudal end of the BDNF-producing cell transplants, although most failed to penetrate the host spinal cord distal to the lesion. These results indicate that whereas fibroblast cell transplants alone can support regrowth of axons from chronically injured supraspinal neurons, modification of these cells to produce BDNF results in a significant increase in the extent of growth into the transplant.     

Protective effects of adenoviral cardiotrophin-1 gene transfer on rubrospinal neurons after spinal cord injury in adult rats

Cardiotrophin-1 (CT-1), a muscle-derived cytokine, supports the survival of motoneurons in vivo and in vitro. The present study investigated whether adenoviral huCT-1 gene transfer protected injured neurons from cell death or atrophy and promoted regeneration of rubrospinal tract (RST) after spinal cord injury in adult rats. Administration of the adenoviral CT-1 vector (Adv-CT1) to C3-4 lateral funiculus hemisection cavity, that completely interrupted RST, led to sustained CT-1 expression. Providing Adv-CT1, which rescued 20% of neurons, could prevent the loss of injured rubrospinal neurons 8 weeks post-injury. Retrograde tracing with FluoroGold showed that 1.2% of RST neurons regenerated at least two segments caudal to the injury site. Anterograde tracing with biotinylated dextran amine revealed that the RST axons terminated in white matter and gray matter. Behavioral testing revealed a significant functional recovery in limb usage. This observation indicated that adenoviral CT-1 gene transfer into the injured cord promoted survival and regeneration of rubrospinal neurons in adult rats.     

Transplantation of genetically modified cells contributes to repair and recovery from spinal injury

The effects of transplantation of fibroblasts genetically modified to produce brain derived neurotrophin factor (Fb/BDNF) on rescue of axotomized neurons, axonal growth and recovery of function was tested in a lateral funiculus lesion model in adult rats. Operated control animals included those in which the lesion was filled with gelfoam implant (Hx) and those in which the cavity was filled with unmodified fibroblasts (Fb). Both Fb/BDNF and Fb transplants survived and filled the lesion site. Unoperated control groups showed a marked retrograde death of Red nucleus neurons contralateral to the lesion; Fb/BDNF recipients showed a significant rescue effect. Anterograde and retrograde labeling studies indicated no regeneration of rubrospinal axons into the lesion/transplant in operated control animals, but regeneration into, around, and through the transplant into the host was seen in the Fb/BDNF recipients. All animals showed deficits on the more challenging behavioral tests but the Fb/BDNF recipients showed fewer deficits, particularly in tests of spontaneous vertical exploration, horizontal rope crossing and a sensory test (patch removal). The improved function on these tests in the Fb/BDNF recipients was abolished by a second lateral funiculus lesion rostral to the transport site. These results indicate that delivery of neurotrophic factors by grafting genetically modified cells can improve repair and function after spinal injury.     

Regenerating descending axons preferentially reroute to the gray matter in the presence of a general macrophage/microglial reaction caudal to a spinal transection in adult zebrafish

We analyzed pathway choices of regenerating, mostly supraspinal, descending axons in the spinal cord of adult zebrafish and the cellular changes in the spinal cord caudal to a lesion site after complete spinal transection. Anterograde tracing (by application of the tracer rostral to the spinal lesion site) showed that significantly more descending axons (74%) regenerated in the spinal gray matter of the caudal spinal cord than would be expected from random growth. Retrograde tracing (by application of the tracer caudal to the spinal lesion site) showed that, rostral to the lesion, most of these axons (80%) extended into the major white matter tracts. Thus, ventral descending tracts often were devoid of labeled axons caudal to a spinal lesion but contained many axons rostral to the lesion in the same animals, indicating a pathway switch of descending axons from the white matter to the gray matter. Ascending axons of spinal neurons were not observed regrowing to the rostral tracer application site; therefore, they most likely did not contribute to the axonal populations analyzed. A macrophage/microglia response within 2 days of spinal cord transection, along with phagocytosis of myelin, was observed caudal to the transection by immunohistochemistry and electron microscopy. Nevertheless, caudal to the lesion, descending tracts in the white matter were filled with myelin debris during the time of axonal regrowth, at least up to 6 weeks postlesion. We suggest that the spontaneous regeneration of axons of supraspinal origin after spinal cord transection in adult zebrafish may be due in part to the axons' ability to negotiate novel pathways in the spinal cord gray matter.     

Delayed grafting of BDNF and NT-3 producing fibroblasts into the injured spinal cord stimulates sprouting, partially rescues axotomized red nucleus neurons from loss and atrophy, and provides limited regeneration

Ex vivo gene therapy, utilizing modified fibroblasts that deliver BDNF or NT-3 to the acutely injured spinal cord, has been shown to elicit regeneration and recovery of function in the adult rat. Delayed grafting into the injured spinal cord is of great clinical interest as a model for treatment of chronic injury but may pose additional obstacles that are not present after acute injury, such as the need to remove an established scar, increased retrograde cell loss and/or atrophy, and diminished capacity for regeneration by neurons which may be doubly injured. The purpose of the present study was to determine if delayed grafting of neurotrophin secreting fibroblasts would have anatomical effects similar to those seen in acute grafting models. We grafted a mixture of BDNF and NT-3 producing fibroblasts or control fibroblasts into a complete unilateral cervical hemisection after a 6-week delay. Fourteen weeks after delayed grafting we found that both the neurotrophin secreting fibroblasts and control fibroblasts survived, but that only the neurotrophin secreting grafts provided a permissive environment for host axon growth, as indicated by immunostaining for RT-97, a marker for axonal neurofilaments, GAP-43, a marker for elongating axons, CGRP, a marker for dorsal root axons, and 5-HT, a marker for raphe spinal axons, within the graft. Anterograde tracing of the uninjured vestibulospinal tract showed growth into neurotrophin producing transplants but not into control grafts, while anterograde tracing of the axotomized rubrospinal tract showed a small number of regenerating axons within the genetically modified grafts, but none in control grafts. The neurotrophin expressing grafts, but not the control grafts, significantly reduced retrograde degeneration and atrophy in the injured red nucleus. Grafts of BDNF + NT-3 expressing fibroblasts delayed 6 weeks after injury therefore elicit growth from intact segmental and descending spinal tracts, stimulate modest regenerative growth by rubrospinal axons, and partially rescue axotomized supraspinal neurons and protect them from atrophy. The regeneration of rubrospinal axons into delayed transplants was much less than has been observed when similar transplants were placed acutely into a lateral funiculus or, after a 4-week delay, into a hemisection lesion. This suggests that the regenerative capacity of chronically injured red nucleus neurons was markedly diminished. The increased GAP43 reactivity in the corticospinal tracts ipsilaterally and contralaterally to the combination grafts suggests that these axons remain responsive to the neurotrophins, that the neurotrophins may stimulate both regenerative and sprouting responses, and that the grafted cells continue to secrete the neurotrophins.     

Undesired effects of a combinatorial treatment for spinal cord injury--transplantation of olfactory ensheathing cells and BDNF infusion to the red nucleus

Transplantations of olfactory ensheathing cells (OECs) have been reported to promote axonal regeneration and functional recovery after spinal cord injury, but have demonstrated limited growth promotion of rat rubrospinal axons after a cervical dorsolateral funiculus crush. Rubrospinal neurons undergo massive atrophy after cervical axotomy and show only transient expression of regeneration-associated genes. Cell body treatment with brain-derived neurotrophic factor (BDNF) prevents this atrophy, stimulates regeneration-associated gene expression and promotes regeneration of rubrospinal axons into peripheral nerve transplants. Here, we hypothesized that the failure of rubrospinal axons to regenerate through a bridge of OEC transplants was due to this weak intrinsic cell body response. Hence, we combined BDNF treatment of rubrospinal neurons with transplantation of highly enriched OECs derived from the nasal mucosa and assessed axonal regeneration as well as behavioral changes after a cervical dorsolateral funiculus crush. Each treatment alone as well as their combination prevented the dieback of the rubrospinal axons, but none of them promoted rubrospinal regeneration beyond the lesion/transplantation site. Motor performance in a food-pellet reaching test and forelimb usage during vertical exploration (cylinder test) were more impaired after combining transplantation of OECs with BDNF treatment. This impaired motor performance correlated with lowered sensory thresholds in animals receiving the combinatorial therapy - which were not seen with each treatment alone. Only this combinatorial treatment group showed enhanced sprouting of calcitonin gene-related peptide-positive axons rostral to the lesion site. Hence, some combinatorial treatments, such as OECs with BDNF, may have undesired effects in the injured spinal cord.     

Treatment of chronically injured spinal cord with neurotrophic factors stimulates betaII-tubulin and GAP-43 expression in rubrospinal tract neurons

Exogenous neurotrophic factors provided at a spinal cord injury site promote regeneration of chronically injured rubrospinal tract (RST) neurons into a peripheral nerve graft. The present study tested whether the response to neurotrophins is associated with changes in the expression of two regeneration-associated genes, betaII-tubulin and growth-associated protein (GAP)-43. Adult female rats were subjected to a right full hemisection lesion via aspiration of the C3 spinal cord. A second aspiration lesion was made 4 weeks later and gel foam saturated in brain-derived neurotrophic factor (BDNF), glial cell-line derived neurotrophic factor (GDNF), or phosphate-buffered saline (PBS) was applied to the lesion site for 60 min. Using in situ hybridization, RST neurons were examined for changes in mRNA levels of betaII-tubulin and GAP-43 at 1, 3, and 7 days after treatment. Based on analysis of gene expression in single cells, there was no effect of BDNF treatment on either betaII-tubulin or GAP-43 mRNA expression at any time point. betaII-Tubulin mRNA levels were enhanced significantly at 1 and 3 days in animals treated with GDNF relative to levels in animals treated with PBS. Treatment with GDNF did not affect GAP-43 mRNA levels at 1 and 3 days, but at 7 days there was a significant increase in mRNA expression. Interestingly, 7 days after GDNF treatment, the mean cell size of chronically injured RST neurons was increased significantly. Although GDNF and BDNF both promote axonal regeneration by chronically injured neurons, only GDNF treatment is associated with upregulation of betaII-tubulin or GAP-43 mRNA. It is not clear from the present study how exogenous BDNF stimulates regrowth of injured axons.     

Regeneration of adult dorsal root axons into transplants of embryonic spinal cord

Transplants of the embryonic rat spinal cord survive and differentiate in the spinal cords of adult and newborn host rats. Very little is known about the extent to which these homotopic transplants can provide an environment for regeneration of adult host axons that normally terminate in the spinal cord. We have used horseradish peroxidase injury filling and transganglionic transport methods to determine whether transected dorsal roots regenerate into fetal spinal cord tissue grafted into the spinal cords of adult rats. Additional transplants were examined for the presence of calcitonin gene-related peptide-like immunoreactivity, which in the normal dorsal horn is derived exclusively from primary afferent axons. Host animals had one side of the L4-5 spinal cord resected and replaced by a transplant of E14 or E15 spinal cord. Adjacent dorsal roots were sectioned and juxtaposed to the graft. The dorsal roots and their projections into the transplants were then labeled 2-9 months later. The tracing methods that used transport or diffusion of horseradish peroxidase demonstrated that severed host dorsal root axons had regenerated and grown into the transplants. In addition, some donor and host neurons had extended their axons into the periphery to at least the midthigh level as indicated by retrograde labeling following application of tracer to the sciatic nerve. Primary afferent axons immunoreactive for calcitonin gene-related peptide were among those that regenerated into transplants, and the projections shown by this immunocytochemical method exceeded those demonstrated by the horseradish peroxidase tracing techniques. Growth of the host dorsal roots into transplants indicates that fetal spinal cord tissue permits regeneration of adult axotomized neurons that would otherwise be aborted at the dorsal root/spinal cord junction. This transplantation model should therefore prove useful in studying the enhancement and specificity of the regrowth of axons that normally terminate in the spinal cord.     

Effects of root replantation and neurotrophic factor treatment on long-term motoneuron survival and axonal regeneration after C7 spinal root avulsion

In order to determine the effect of nerve root replantation on motoneuron survival and regeneration, we have avulsed and replanted C7 ventral rootlets in adult rabbits under various conditions. Intraspinal alterations and exact positions of ventrolateral replantations were studied in each animal, and the effects of BDNF and/or CNTF administration during replantation investigated in different experimental groups. Six months after lesion, about 70% of motoneurons were lost on the lesioned sides in the C7 segment, without significant differences between groups. Retrograde fluorescent tracing and histological analysis documented that many axons had regrown through the original ventral exit zones or had exited the spinal cord at the lateral replantation site. However, many laterally exiting axons had not grown out directly from the ventral horn through the lateral white matter but had elongated vertically before leaving the spinal cord. The mean axonal diameter was significantly higher in regenerated axons that had exited through the original ventral exit zones in comparison with axons which had grown out laterally. Application of BDNF and/or CNTF did not show any effects on the pathways of regeneration into the replanted root. The results indicate that motoneuron survival cannot be significantly improved by a single dose of neurotrophic factors applied to a ventrolateral replantation site. However, a significant number of myelinating axons are found in replanted roots, and regeneration may be more efficient when outgrowth through the original ventral exit zone is supported.     

Regeneration of nigrostriatal dopaminergic axons after transplantation of olfactory ensheathing cells and fibroblasts prevents fibrotic scar formation at the lesion site

The fibrotic scar formed after central nervous system injury has been considered an obstacle to axonal regeneration. The present study was designed to examine whether cell transplantation into a damaged central nervous system can reduce fibrotic scar formation and promote axonal regeneration. Nigrostriatal dopaminergic axons were unilaterally transected in rats and cultures of olfactory-ensheathing cells (OECs), and olfactory nerve fibroblasts were transplanted into the lesion site. In the absence of transplants, few tyrosine hydroxylase-immunoreactive axons extended across the lesion 2 weeks after the transection. Reactive astrocytes increased around the lesion, and a fibrotic scar containing type IV collagen deposits developed in the lesion center. The immunoreactivity of chondroitin sulfate side chains and core protein of NG2 proteoglycan increased in and around the lesion. One and 2 weeks after transection and simultaneous transplantation, dopaminergic axons regenerated across the transplanted tissues, which consisted of p75-immunoreactive OECs and fibronectin-immunoreactive fibroblasts. Reactive astrocytes and chondroitin sulfate immunoreactivity increased around the transplants, whereas the deposition of type IV collagen and fibrotic scar formation were completely prevented at the lesion site. Transplantation of meningeal fibroblasts similarly prevented the formation of the fibrotic scar, although its effect on regeneration was less potent than transplantation of OECs and olfactory nerve fibroblasts. The present results suggest that elimination of the inhibitory fibrotic scar is important for neural regeneration.     

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.     

Spinal cord transplants permit the growth of serotonergic axons across the site of neonatal spinal cord transection

These experiments were designed to determine whether transplants of fetal spinal cord tissue into lesioned spinal cord in newborn rats provide a terrain that supports the growth of serotonergic (5-HT) axons across the site of the lesion. Although descending serotonergic axons can regenerate after chemical lesions in adult animals, they show little regrowth after surgical lesions. In newborn animals, 5-HT axons do not regrow after either chemical or mechanical lesions since the axotomized raphe-spinal neurons die. After partial spinal cord lesions made in developing animals, immature axons can take an aberrant route around the site of the lesion to reach normal target areas. Even these robust, late-growing, uninjured axons, however, are unable to grow through the site of the spinal cord lesion. Immunocytochemical labeling was used to determine if descending serotonergic axons grow into fetal spinal cord transplants, and whether these axons cross the transplant to reach spinal cord levels caudal to the lesion. Spinal cord transection at a mid-thoracic spinal cord level on the day of birth resulted in a dramatic decrease in 5-HT immunoreactivity caudal to the lesion by one day postoperative. 5-HT immunoreactivity caudal to the lesion was abolished by 5 days postoperative and did not return after acute or chronic (6 months) survival periods. When a transplant of fetal spinal cord tissue was placed into the lesion site, 5-HT axons were identified throughout the transplant. At spinal cord levels caudal to the transection and transplant, the serotonergic axons were identified in the host spinal cord in both the white and gray matter. This 5-HT innervation was not confined to spinal cord segments adjacent to the lesion site but extended to spinal cord segments as far as lower lumbar levels. The reinnervation of the host spinal cord caudal to the transection was far less than that seen in unlesioned adult rat spinal cord. Horseradish peroxidase (HRP) injected caudal to the transection and transplant, retrogradely labeled neurons within the medullary raphe nuclei. The HRP and 5-HT results both depended on apposition of the transplant with the rostral and caudal stumps of the host spinal cord; without such apposition, labeling was abolished. These results indicate that the presence of a transplant at the site of the neonatal lesion modifies the environment at the lesion site in such a manner as to support the elongation of identified axons across the site of the lesion and into the host cord caudal to the lesion.     

NT-3 promotes growth of lesioned adult rat sensory axons ascending in the dorsal columns of the spinal cord

The regeneration capacity of spinal cord axons is severely limited. Recently, much attention has focused on promoting regeneration of descending spinal cord pathways, but little is known about the regenerative capacity of ascending axons. Here we have assessed the ability of neurotrophic factors to promote regeneration of sensory neurons whose central axons ascend in the dorsal columns. The dorsal columns of adult rats were crushed and either brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), neurotrophin-3 (NT-3) or a vehicle solution was delivered continuously to the lesion site for 4 weeks. Transganglionic labelling with cholera toxin beta subunit (CTB) was used to selectively label large myelinated Abeta fibres. In lesioned rats treated with vehicle, CTB-labelled fibres were observed ascending in the gracile fasciculus, but these stopped abruptly at the lesion site, with no evidence of sprouting or growth into lesioned tissue. No CTB-labelled terminals were observed in the gracile nucleus, indicating that the lesion successfully severed all ascending dorsal column axons. Treatment with BDNF did not promote axonal regeneration. In GDNF-treated rats fibres grew around cavities in caudal degenerated tissue but did not approach the lesion epicentre. NT-3, in contrast, had a striking effect on promoting growth of lesioned dorsal column axons with an abundance of fibre sprouting apparent at the lesion site, and many fibres extending into and beyond the lesion epicentre. Quantification of fibre growth confirmed that only in NT-3-treated rats did fibres grow into the crush site and beyond. No evidence of terminal staining in the gracile nucleus was apparent following any treatment. Thus, although NT-3 promotes extensive growth of lesioned axons, other factors may be required for complete regeneration of these long ascending projections back to the dorsal column nuclei. The intrathecal delivery of NT-3 or other neurotrophic molecules has obvious advantages in clinical applications, as we show for the first time that dorsal column axonal regeneration can be achieved without the use of graft implantation or nerve lesions.     

Sensory afferents regenerated into dorsal columns after spinal cord injury remain in a chronic pathophysiological state

Axon regeneration after experimental spinal cord injury (SCI) can be promoted by combinatorial treatments that increase the intrinsic growth capacity of the damaged neurons and reduce environmental factors that inhibit axon growth. A prior peripheral nerve conditioning lesion is a well-established means of increasing the intrinsic growth state of sensory neurons whose axons project within the dorsal columns of the spinal cord. Combining such a prior peripheral nerve conditioning lesion with the infusion of antibodies that neutralize the growth inhibitory effects of the NG2 chondroitin sulfate proteoglycan promotes sensory axon growth through the glial scar and into the white matter of the dorsal columns. The physiological properties of these regenerated axons, particularly in the chronic SCI phase, have not been established. Here we examined the functional status of regenerated sensory afferents in the dorsal columns after SCI. Six months post-injury, we located and electrically mapped functional sensory axons that had regenerated beyond the injury site. The regenerated axons had reduced conduction velocity, decreased frequency-following ability, and increasing latency to repetitive stimuli. Many of the axons that had regenerated into the dorsal columns rostral to the injury site were chronically demyelinated. These results demonstrate that regenerated sensory axons remain in a chronic pathophysiological state and emphasize the need to restore normal conduction properties to regenerated axons after spinal cord injury.     

Retinal ganglion cell and nonneuronal cell responses to a microcrush lesion of adult rat optic nerve

Injury of the optic nerve has served as an important model for the study of cell death and axon regeneration in the CNS. Analysis of axon sprouting and regeneration after injury by anatomical tracing are aided by lesion models that produce a well-defined injury site. We report here the characterization of a microcrush lesion of the optic nerve made with 10-0 sutures to completely transect RGC axons. Following microcrush lesion, 62% of RGCs remained alive 1 week later, and 28% of RGCs, at 2 weeks. Optic nerve sections stained by hematoxylin-based methods showed a thin line of intensely stained cells that invaded the lesion site at 24 h after microcrush lesion. The lesion site became increasingly disorganized by 2 weeks after injury, and both macrophages and blood vessels invaded the lesion site. The microcrush lesion was immunoreactive for chondroitin sulfate proteoglycans (CSPG), and an adjacent GFAP-negative zone developed early after the lesion, disappearing by 1 week. Luxol fast blue staining showed a myelin-free zone at the lesion site, and myelin remained distal to the lesion at 8 weeks. To study the axonal response to microcrush lesion, anterograde tracing was used. Within 6 h after injury all RGC axons retracted back from the site of lesion. By 1 week after injury, axons regrew toward the lesion, but most stopped abruptly at the injury scar. The few axons that were able to cross the injury site did not extend further in the optic nerve white matter by 8 weeks postlesion. Our observations suggest that both the CSPG-positive scar and the myelin-derived growth inhibitory proteins contribute to the failure of RGC regeneration after injury.     

Human adult olfactory neural progenitors promote axotomized rubrospinal tract axonal reinnervation and locomotor recovery

We investigated the effects of engrafted human adult olfactory neuroepithelial neurosphere forming cells (NSFCs) on regeneration and reinnervation of rubrospinal tract (RST) axons and locomotor recovery following partial cervical hemisection that completely ablated the RST. Weekly behavioral testing showed greater functional recovery of forelimb use during the 12 weeks after NSFCs engraftment than in the control rats. Anterograde tracing with biotinylated dextran amine (BDA) confirmed the presence of RST axons within the white matter 4-8 segments caudal to the grafts. Both immunofluorescence and immunoelectron microscopy revealed the BDA-labeled RST axonal terminals reestablished synaptic connections with motoneurons in the ventral horn of the distal cervical spinal cord. Further study of forelimb functional recovery in NSFCs-engrafted subgroups considered the effects of a second dorsolateral funiculotomy, irreversibly destroying the recovery, and the injection of muscimol, blocking RST neuronal activity reversibly. These results highlight the unique potential of human olfactory neuroepithelial-derived progenitors as an autologous cell source for spinal cord repair.