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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 fetus-derived 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., (Copyright 2002 Wiley-Liss, Inc.)

The responses of the central (CNS) and peripheral (PNS) nervous system to axotomy differ in a number of ways; these differences can be observed in both the cell body responses to injury and in the extent of regeneration that occurs in each system. The cell body responses to injury in the PNS involves the upregulation of genes that are not upregulated following comparable injuries to CNS neurons. The expression of particular genes following injury may be essential for regeneration to occur. In the present study, we have evaluated the hypothesis that expression of the inducible transcription factor c-Jun is associated with regrowth of axotomized CNS neurons. In these experiments, we compared c-Jun expression in axotomized brainstem neurons after thoracic spinal cord hemisection alone (a condition in which no regrowth occurs) and in groups of animals where hemisections were combined with treatments such as transplants of fetal spinal cord tissue and/or application of neurotrophic factors to the lesion site. The latter conditions enhance the capacity of the CNS for regrowth. We have demonstrated that hemisections alone do not upregulate expression of c-Jun, indicating that this particular cell body response is not a direct result of axotomy. However, c-Jun expression is upregulated in animals that received application of transplants and neurotrophins. Because these interventions also promote sprouting and regrowth of CNS axons after spinal cord lesions, we suggest that transplants and exogenous neurotrophic factor application activate a cell body response consistent with a role for c-Jun in axonal growth., (Copyright 1999 Academic Press.)

Neonatal midthoracic spinal cord injury disrupts the development of postural reflexes and hindlimb locomotion. The recovery of rhythmical alternating movements, such as locomotion, is enhanced in injured animals receiving fetal spinal cord transplants. Neonatal cervical spinal cord injury disrupts not only locomotion but also skilled forelimb movement. The aims of this study were to determine the consequences of cervical spinal cord injury on forelimb motor function and to determine whether transplants of fetal spinal cord support normal development of skilled forelimb use after this injury. Three-day-old rats received a cervical spinal cord lesion at C3, with or without a transplant of fetal cervical spinal cord (embryonic day 14); unoperated pups served as controls. Animals were examined daily during the first month of life using a behavioral protocol that assessed reflexes, postural reactions, and forelimb motor skills. They also were trained and tested as adults to assess performance in goal-directed reaching tasks. The onset of postural reflexes was delayed in the lesion-only group, and goal-directed reaching and associated postural adjustments failed to develop. The transplant group developed reflex responses and skilled forelimb activity that resembled normal movement patterns. Transplant animals developed both target reaching and accompanying postural adjustments. Target reaching requires integration of segmental, intersegmental, and supraspinal input to propriospinal and motor neurons over many spinal cord levels. Transplants may support the reestablishment of input onto these neurons, permitting the development of skilled forelimb activity after neonatal cervical spinal cord injury. The neuroanatomical reorganization of descending and propriospinal input was examined in the companion paper (Diener and Bregman, 1998).

Cervical spinal cord injury at birth permanently disrupts forelimb function in goal-directed reaching. Transplants of fetal spinal cord tissue permit the development of skilled forelimb use and associated postural adjustments (, companion article). The aim of this study was to determine whether transplants of fetal spinal cord tissue support the remodeling of supraspinal and segmental pathways that may underlie recovery of postural reflexes and forelimb movements. Although brainstem-spinal and segmental projections to the cervical spinal cord are present at birth, skilled forelimb reaching has not yet developed. Three-day-old rats received a cervical spinal cord overhemisection with or without transplantation of fetal spinal cord tissue (embryonic day 14); unoperated pups served as normal controls. Neuroanatomical tracing techniques were used to examine the organization of CNS pathways that may influence target-directed reaching. In animals with hemisections only, corticospinal, brainstem-spinal, and dorsal root projections within the spinal cord were decreased in number and extent. In contrast, animals receiving hemisections plus transplants exhibited growth of these projections throughout the transplant and over long distances within the host spinal cord caudal to the transplant. Raphespinal axons were apposed to numerous propriospinal neurons in control and transplant animals; these associations were greatly reduced in the lesion-only animals. These observations suggest that after neonatal cervical spinal cord injury, embryonic transplants support axonal growth of CNS pathways and specifically supraspinal input to propriospinal neurons. We suggest that after neonatal spinal injury in the rat, the transplant-mediated reestablishment of supraspinal input to spinal circuitry is the mechanism underlying the development of target-directed reaching and associated postural adjustments.

Axotomy of mature rubrospinal neurons leads to a substantial atrophy of these neurons within days after surgery. In addition, these neurons do not successfully regenerate following axotomy. The relationship of atrophy to regenerative failure is not clear, and the signals which regulate these events have not been identified. However, it is possible that the atrophy of these neurons plays a role in preventing regeneration. In the present study, we evaluated the hypothesis that interventions which have been shown to promote growth of axotomized CNS neurons are also capable of reversing the axotomy-induced atrophy. To test this hypothesis, adults rats received thoracic spinal cord hemisection alone or in combination with transplants of fetal spinal cord tissue and/or neurotrophic factor support. Our data indicate that application of either transplants or neurotrophic factors partially reverse the axotomy-induced atrophy in rubrospinal neurons, but that both interventions together reverse the atrophy completely. These results suggest that the same pathways that are activated to enhance growth of rubrospinal neurons after axotomy may also be involved in the maintenance of cell morphology.

The capacity of CNS neurons for axonal regrowth after injury decreases as the age of the animal at time of injury increases. After spinal cord lesions at birth, there is extensive regenerative growth into and beyond a transplant of fetal spinal cord tissue placed at the injury site. After injury in the adult, however, although host corticospinal and brainstem-spinal axons project into the transplant, their distribution is restricted to within 200 micron of the host/transplant border. The aim of this study was to determine if the administration of neurotrophic factors could increase the capacity of mature CNS neurons for regrowth after injury. Spinal cord hemisection lesions were made at cervical or thoracic levels in adult rats. Transplants of E14 fetal spinal cord tissue were placed into the lesion site. The following neurotrophic factors were administered at the site of injury and transplantation: brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), neurotrophin-4 (NT-4), ciliary-derived neurotrophic factor (CNTF), or vehicle alone. After 1-2 months survival, neuroanatomical tracing and immunocytochemical methods were used to examine the growth of host axons within the transplants. The neurotrophin administration led to increases in the extent of serotonergic, noradrenergic, and corticospinal axonal ingrowth within the transplants. The influence of the administration of the neurotrophins on the growth of injured CNS axons was not a generalized effect of growth factors per se, since the administration of CNTF had no effect on the growth of any of the descending CNS axons tested. These results indicate that in addition to influencing the survival of developing CNS and PNS neurons, neurotrophic factors are able to exert a neurotropic influence on injured mature CNS neurons by increasing their axonal growth within a transplant., (Copyright 1997 Academic Press.)

After spinal cord injury at birth, axotomized brainstem-spinal and corticospinal neurons are capable of permanent regenerative axonal growth into and through a fetal spinal cord transplant placed into the site of either a spinal cord hemisection or transection. In contrast, if fetal tissue which is not a normal target of the axotomized neurons (embryonic hippocampus or cortex) is placed into a neonatal spinal cord hemisection, brainstem-spinal serotonergic axons transiently innervate the transplant, but subsequently withdraw. The first set of experiments was designed to test the hypothesis that after spinal cord transection, serotonergic axons would cross the nontarget transplant, reach normal spinal cord targets caudal to the transection, and gain access to requisite target-derived cues, permitting permanent maintenance. Surprisingly, after a complete spinal cord transection, brainstem-spinal axons failed to grow into an inappropriate target even transiently. These observations suggest that the transient axonal ingrowth into nontarget transplants may represent lesion-induced axonal sprouting by contralateral uninjured axons. We have used double-labeling with fluorescent dyes, to test directly whether axonal sprouting of neurons which maintain collaterals to uninjured spinal cord targets (1) provide the transient ingrowth of brainstem-spinal axons into a nontarget transplant and (2) contribute to permanent ingrowth into target-specific transplants. Uninjured red nucleus, raphe nucleus, and locus coeruleus neurons extend axons into the nontarget transplant while maintaining collaterals to the host spinal cord caudal to the transplant. The lesion-induced sprouting by uninjured axons was also observed with a target-specific transplant. Taken together, these studies suggest that sprouting and regenerating axons may differ in their requirements for growth after injury.

Neurons that maintain extensive axon collaterals proximal to the site of axotomy may be better able to survive injury. Early lesions of the rubrospinal tract lead to retrograde cell death of the majority of axotomized immature neurons. Transplants of fetal spinal cord tissue rescue axotomized rubrospinal neurons and promote their axonal regeneration. Rubrospinal neurons develop many of their axon collaterals postnatally. The present study tests the hypothesis that the axotomized rubrospinal neurons that are rescued by transplants and regenerate their axons are those neurons that have established axon collaterals to targets rostral to the lesion. Neonatal rats received a transplant of fetal spinal cord tissue placed into a midthoracic spinal cord hemisection. One month after transplantation, the retrogradely transported fluorescent tracers fast blue (FB) and diamidino yellow (DY) were used to identify rubrospinal neurons with collaterals to particular targets. FB was injected either into the interpositus nucleus of the cerebellum or into the gray matter of the cervical enlargement to identify collaterals to these targets, and DY was injected into the spinal cord approximately 5 mm caudal to the transplant and lesion site to label retrogradely the neurons that regenerated their axons. Double labeling was observed in the axotomized neurons of the red nucleus after tracer injections into the cervical spinal cord but not after injections into the cerebellum. This labeling pattern indicates that axotomized rubrospinal neurons that are rescued and regenerate axons caudal to the transplant maintain axon collaterals at cervical spinal cord levels. Cerebellar collaterals do not appear to play a role in the survival and regrowth of axotomized rubrospinal neurons.

The response of the mature central nervous system (CNS) to injury differs significantly from the response of the peripheral nervous system (PNS). Axotomized PNS neurons generally regenerate following injury, while CNS neurons do not. The mechanisms that are responsible for these differences are not completely known, but both intrinsic neuronal and extrinsic environmental influences are likely to contribute to regenerative success or failure. One intrinsic factor that may contribute to successful axonal regeneration is the induction of specific genes in the injured neurons. In the present study, we have evaluated the hypothesis that expression of the immediate early gene c-jun is involved in a successful regenerative response. We have compared c-Jun expression in dorsal root ganglion (DRG) neurons following central or peripheral axotomy. We prepared animals that received either a sciatic nerve (peripheral) lesion or a dorsal rhizotomy in combination with spinal cord hemisection (central lesion). In a third group of animals, several dorsal roots were placed into the hemisection site along with a fetal spinal cord transplant. This intervention has been demonstrated to promote regrowth of severed axons and provides a model to examine DRG neurons during regenerative growth after central lesion. Our results indicated that c-Jun was upregulated substantially in DRG neurons following a peripheral axotomy, but following a central axotomy, only 18% of the neurons expressed c-Jun. Following dorsal rhizotomy and transplantation, however, c-Jun expression was upregulated dramatically; under those experimental conditions, 63% of the DRG neurons were c-Jun-positive. These data indicate that c-Jun expression may be related to successful regenerative growth following both PNS and CNS lesions.

After spinal cord lesions in newborn rats, transplants of fetal spinal cord tissue rescue immature axotomized neurons, support the growth of axons into and through the site of injury and prolong the critical period for developmental plasticity. Both late-developing (uninjured) and regenerating axons contribute to this transplant-induced anatomical plasticity. After lesions in the mature CNS, transplant-induced axonal elongation is spatially restricted. The current study was designed (1) to determine the magnitude of transplant-induced regeneration, (2) to test the hypothesis that the long distance growth beyond the site of injury is mediated by late-developing axonal pathways, whereas axonal elongation by regenerating pathways is spatially restricted as it is in the adult, and (3) to determine if particular nuclei have a greater inherent capacity for regeneration than others. We used temporally spaced retrograde tracing with the fluorescent dyes fast blue and diamidino yellow to address this issue. Fast blue was placed into the site of a spinal cord overhemisection in rat pups < 48 h old to label those neurons which were axotomized by a neonatal lesion. The tracer was removed and a transplant of Embryonic Day 14 fetal spinal cord tissue was placed into the lesion site. Three to six weeks later a second tracer (diamidino yellow) was injected bilaterally into the host spinal cord caudal to the transplant. We counted the number of double-labeled (regenerated), single diamidino yellow-labeled (late-growing), and single fast blue-labeled (nonbridging) neurons in the cortex, red nucleus, raphe nuclei, and locus coeruleus. By systematically varying the distance of the diamidino yellow injection site caudal to the transplant, we were able to compare the distance which injured axons regenerate with the distance that late-growing axons extend. When the diamidino yellow injection was placed within 5 mm caudal to the transplant 28% of the axotomized neurons in the red nucleus, 32% of the axotomized neurons in the locus coeruleus, and 37% of the axotomized neurons in the raphe nuclei were double-labeled (regenerating). Although the percentage of double-labeled neurons decreased as the distance beyond the transplant increased, a substantial population of regenerating neurons was identified in each of the brain stem nuclei examined following diamidino yellow injections up to 15 mm caudal to the transplant. Thus, after spinal cord lesions and transplants at birth, both regenerating neurons and late-developing neurons extended axons long distances (up to 15 m) caudal to the lesion site. The capacity for regenerative growth was similar in each of the nuclei examined.

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.

Fetal spinal cord transplants prevent the retrograde cell death of immature axotomized central nervous system (CNS) neurons and provide a terrain which supports axonal elongation in the injured immature spinal cord. The current experiments were designed to determine whether the axons which grow across the site of the neonatal lesion and transplant are derived from axotomized neurons and are therefore regenerating or whether the axons which grow across the transplant are late-growing axons that have not been axotomized directly. We have used an experimental paradigm of midthoracic spinal cord lesion plus transplant at birth and temporally spaced retrograde tracing with the fluorescent tracers fast blue (FB) and diamidino yellow (DY) to address this issue. Fast blue was placed into the site of a spinal cord hemisection in rat pups less than 48 h old. After 3-6 h to allow uptake and transport of the tracer, the source of fast blue was removed by aspiration and the lesion was enlarged to an "over-hemisection." A transplant of Embryonic Day 14 fetal spinal cord tissue was placed into the lesion site. The animals survived 3-6 weeks prior to the injection of the second tracer (DY) bilaterally into the host spinal cord caudal to the lesion plus transplant. Neurons with late-developing axons would not be exposed to the first dye (FB), but could only be exposed to the second tracer, diamidino yellow. Thus, neurons with a diamidino yellow-labeled nucleus are interpreted as "late-developing" neurons. Neurons axotomized by midthoracic spinal cord lesion at birth could be exposed to the first tracer, fast blue. If after axotomy they regrew caudal to the transplant, they could be labeled by the second tracer as well. We interpret these double-labeled neurons as regenerating neurons. If neurons labeled with fast blue and axotomized by the spinal cord hemisection either failed to regenerate or grew into the transplant but not caudal to it, they would be labeled only by the first dye. We have examined the pattern and distribution of single (FB or DY)- and double (FB + DY)-labeled neurons in the sensorimotor cortex, red nucleus, locus coeruleus, and raphe nuclei. The sensorimotor cortex contains only DY-labeled neurons. The red nucleus contains both FB- and FB + DY-labeled neurons.(ABSTRACT TRUNCATED AT 400 WORDS)