Transplants of cells genetically modified to express neurotrophin-3 rescue axotomized Clarke's nucleus neurons after spinal cord hemisection in adult rats.

To test the idea that genetically engineered cells can rescue axotomized neurons, we transplanted fibroblasts and immortalized neural stem cells (NSCs) modified to express neurotrophic factors into the injured spinal cord. The neurotrophin-3 (NT-3) or nerve growth factor (NGF) transgene was introduced into these cells using recombinant retroviral vectors containing an internal ribosome entry site (IRES) sequence and the beta-galactosidase or alkaline phosphatase reporter gene. Bioassay confirmed biological activity of the secreted neurotrophic factors. Clarke's nucleus (CN) axons, which project to the rostral spinal cord and cerebellum, were cut unilaterally in adult rats by T8 hemisection. Rats received transplants of fibroblasts or NSCs genetically modified to express NT-3 or NGF and a reporter gene, only a reporter gene, or no transplant. Two months postoperatively, grafted cells survived at the hemisection site. Grafted fibroblasts and NSCs expressed a reporter gene and immunoreactivity for the NGF or NT-3 transgene. Rats receiving no transplant or a transplant expressing only a reporter gene showed a 30% loss of CN neurons in the L1 segment on the lesioned side. NGF-expressing transplants produced partial rescue compared with hemisection alone. There was no significant neuron loss in rats receiving grafts of either fibroblasts or NSCs engineered to express NT-3. We postulate that NT-3 mediates survival of CN neurons through interaction with trkC receptors, which are expressed on CN neurons. These results support the idea that NT-3 contributes to long-term survival of axotomized CN neurons and show that genetically modified cells rescue axotomized neurons as efficiently as fetal CNS transplants.

Characterization and intraspinal grafting of EGF/bFGF-dependent neurospheres derived from embryonic rat spinal cord.

Recent advances in the isolation and characterization of neural precursor cells suggest that they have properties that would make them useful transplants for the treatment of central nervous system disorders. We demonstrate here that spinal cord cells isolated from embryonic day 14 Sprague-Dawley and Fischer 344 rats possess characteristics of precursor cells. They proliferate as undifferentiated neurospheres in the presence of EGF and bFGF and can be maintained in vitro or frozen, expanded and induced to differentiate into both neurons and glia. Exposure of these cells to serum in the absence of EGF and bFGF promotes differentiation into astrocytes; treatment with retinoic acid promotes differentiation into neurons. Spinal cord cells labeled with a nuclear dye or a recombinant adenovirus vector carrying the lacZ gene survive grafting into the injured spinal cord of immunosuppressed Sprague-Dawley rats and non-immunosuppressed Fischer 344 rats for up to 4 months following transplantation. In the presence of exogenously supplied BDNF, the grafted cells differentiate into both neurons and glia. These spinal cord cell grafts are permissive for growth by several populations of host axons, especially when combined with exogenous BDNF administration, as demonstrated by penetration into the graft of axons immunopositive for 5-HT and CGRP. Thus, precursor cells isolated from the embryonic spinal cord of rats, expanded in culture and genetically modified, are a promising type of transplant for repair of the injured spinal cord.

Axonal transport of microtubule-associated protein 1B (MAP1B) in the sciatic nerve of adult rat: distinct transport rates of different isoforms.

Cytoskeletal proteins are axonally transported with slow components a and b (SCa and SCb). In peripheral nerves, the transport velocity of SCa, which includes neurofilaments and tubulin, is 1-2 mm/d, whereas SCb, which includes actin, tubulin, and numerous soluble proteins, moves as a heterogeneous wave at 2-4 mm/d. We have shown that two isoforms of microtubule-associated protein 1B (MAP1B), which can be separated on SDS polyacrylamide gels on the basis of differences in their phosphorylation states (band I and band II), were transported at two different rates. All of band I MAP1B moved as a coherent wave at a velocity of 7-9 mm/d, distinct from slow axonal transport components SCa and SCb. Several other proteins were detected within the component that moved at the velocity of 7-9 mm/d, including the leading wave of tubulin and actin. The properties of this component define a distinct fraction of the slow axonal transport that we suggest to term slow component c (SCc). The relatively fast transport of the phosphorylated MAP1B isoform at 7-9 mm/d may account for the high concentration of phosphorylated MAP1B in the distal end of growing axons. In contrast to band I MAP1B, the transport profile of band II was complex and contained components moving with SCa and SCb and a leading edge at SCc. Thus, MAP1B isoforms in different phosphorylation states move with distinct components of slow axonal transport, possibly because of differences in their abilities to associate with other proteins.

Intraspinal delivery of neurotrophin-3 using neural stem cells genetically modified by recombinant retrovirus.

Neural stem cells have been shown to participate in the repair of experimental CNS disorders. To examine their potential in spinal cord repair, we used retroviral vectors to genetically modify a clone of neural stem cells, C17, to overproduce neurotrophin-3 (NT-3). The cells were infected with a retrovirus construct containing the NT-3.IRES.lacZ/neo sequence and cloned by limiting dilution and selection for lacZ expression. We studied the characteristics of the modified neural stem cells in vitro and after transplantation into the intact spinal cord of immunosuppressed adult rats. Our results show that: (i) most of the genetically modified cells express both NT-3 and lacZ genes with a high coexpression ratio in vitro and after transplantation; and (ii) large numbers of the xenografted cells survive in the spinal cord of adult rats for at least 2 months, differentiate into neuronal and glial phenotypes, and migrate for long distances. We conclude that genetically modified neural stem cells, acting as a source of neurotrophic factors, have the potential to participate in spinal cord repair.

Transplants of fibroblasts genetically modified to express BDNF promote regeneration of adult rat rubrospinal axons and recovery of forelimb function.

Adult mammalian CNS neurons do not normally regenerate their severed axons. This failure has been attributed to scar tissue and inhibitory molecules at the injury site that block the regenerating axons, a lack of trophic support for the axotomized neurons, and intrinsic neuronal changes that follow axotomy, including cell atrophy and death. We studied whether transplants of fibroblasts genetically engineered to produce brain-derived neurotrophic factor (BDNF) would promote rubrospinal tract (RST) regeneration in adult rats. Primary fibroblasts were modified by retroviral-mediated transfer of a DNA construct encoding the human BDNF gene, an internal ribosomal entry site, and a fusion gene of lacZ and neomycin resistance genes. The modified fibroblasts produce biologically active BDNF in vitro. These cells were grafted into a partial cervical hemisection cavity that completely interrupted one RST. One and two months after lesion and transplantation, RST regeneration was demonstrated with retrograde and anterograde tracing techniques. Retrograde tracing with fluorogold showed that approximately 7% of RST neurons regenerated axons at least three to four segments caudal to the transplants. Anterograde tracing with biotinylated dextran amine revealed that the RST axons regenerated through and around the transplants, grew for long distances within white matter caudal to the transplant, and terminated in spinal cord gray matter regions that are the normal targets of RST axons. Transplants of unmodified primary fibroblasts or Gelfoam alone did not elicit regeneration. Behavioral tests demonstrated that recipients of BDNF-producing fibroblasts showed significant recovery of forelimb usage, which was abolished by a second lesion that transected the regenerated axons.

Intraspinal grafting of fibroblasts genetically modified by recombinant adenoviruses.

Intracerebral or intraspinal grafting of genetically modified primary fibroblasts has been shown to enhance functional recovery in several models of CNS disease, including spinal cord injury. Most of these studies utilized retrovirus vectors. In this report, we describe in vitro conditions for genetically modifying primary fibroblasts with recombinant adenovirus vectors carrying the lacZ or green fluorescent protein (GFP) genes. As intraspinal allografts in animals immunosuppressed by cyclosporin A, the genetically modified cells survived and expressed the transgenes for at least 2 months. We conclude that recombinant adenovirus vectors are efficient and convenient tools for ex vivo gene therapy in the CNS.

Critical interval for rescue of axotomized neurons by transplants.

To determine whether embryonic spinal cord transplants retained the ability to prevent retrograde death of Clarke's nucleus (CN) neurons if supplied after a delay, we hemisected adult rats at the T8 spinal cord segment and placed transplants of fetal tissue into the hemisection cavity immediately or up to 14 days later. Transplants provided in the first 7 days after injury prevented virtually all of the 30% loss of CN neurons at L1 ipsilateral to hemisection that occurs without a transplant. Transplants supplied at 14 days post-hemisection were ineffective. Because prevention of retrograde neuron death is one mechanism by which transplants may contribute to locomotor recovery after spinal cord injury, this window of effectiveness should be considered in the design of clinical trials.

Neurotrophin-3 prevents death of axotomized Clarke's nucleus neurons in adult rat.

In the present investigation, we studied whether neurotrophin-3 (NT-3) contributes to the rescue of axotomized Clarke's nucleus (CN) neurons in adult rats. A significant (24%) loss of CN neurons occurred at L-1 ipsilateral to T-8 hemisection by 14 days, which reached 31% at 2 months and then stabilized. Axotomized CN neurons had also atrophied by 14 days, but mean cell size did not decrease further. Animals that received gelfoam soaked in nerve growth factor, brain derived neurotrophic factor, or ciliary neurotrophic factor at the lesion site also showed a 30% neuron loss at 2 months, and a 40% reduction in average cell area. Rats receiving NT-3 showed a 15% neuron loss, which was not improved by additional neurotrophins in combination with NT-3. None of the treatments prevented neuron atrophy. Bioassay of the gelfoam showed that NT-3 bioactivity remained at 5 days after surgery but not at 14 days. Additional rats with hemisections that received NT-3 continuously via mini-pump for 2 months showed a 15% neuron loss, the same as with NT-3 given via gelfoam. These results indicate that even limited exposure of axotomized CN neurons to NT-3 produces permanent rescue of 50% of the neurons. The virtually complete rescue that we had previously observed with transplants of fetal central nervous system (CNS) tissues may, therefore, be due at least in part to NT-3, but the exogenous administration of a single neurotrophic factor or a combination of neurotrophic factors is less effective than transplants in producing long-term survival of axotomized CNS neurons.

Application of recombinant adenovirus for in vivo gene delivery to spinal cord.

One strategy for treating spinal cord injury is to supply damaged neurons with the appropriate neurotrophins either by direct delivery or by transfer of the corresponding genes using viral vectors. Here we report the feasibility of using recombinant adenovirus for in vivo gene transfer in spinal cord. After injection of a recombinant adenovirus carrying a beta-galactosidase (beta-gal) reporter gene into the mid-thoracic spinal cord of adult rats, transgene expression occurred not only in several types of cells around the injection site but also in neurons whose axons project to this region from rostral or caudal to the injection site. Among labeled neurons were those of the red nucleus, the vestibular nuclei, reticular formation, locus coeruleus, and Clarke's nucleus. A non-specific immune reaction, which could be blocked by immunosuppression with Cyclosporin A, reduced the number of transduced cells surviving at the injection site by 1 month. In neurons away from the injection site, where the immune response was minimal, transgene expression lasted for at least 2 months. These results support the idea that recombinant adenovirus can be used in the spinal cord for in vivo delivery of therapeutic genes important for supporting neuron survival and axon regeneration.

Embryonic spinal cord transplants enhance locomotor performance in spinalized newborn rats.

The results of the present experiments demonstrate that fetal spinal cord transplants placed into the site of a complete transection in newborn rats permit the development of complex patterns of locomotion. These patterns differ in some respects from normal, but include weight support, appropriate postural adjustment, and coordination between forelimbs and hindlimbs. 5-HT agonists administered to transplanted rats can further modify these motor patterns in ways that may prove able to enhance locomotion. When placed into lesion cavities in adult spinal cord, cells genetically modified to express neurotrophins can survive, differentiate, and mimic at least one consequence of fetal transplants, rescue of axotomized neurons from retrograde cell death.

Fetal spinal cord transplants rescue some axotomized rubrospinal neurons from retrograde cell death in adult rats.

Intraspinal transplants of fetal spinal cord may contribute to recovery after spinal cord injury by keeping axotomized neurons alive. In this study we examined whether transplants rescued axotomized red nucleus (RN) neurons from retrograde cell death in adult rats. RN neurons were labeled by retrograde transport of Fluorogold (FG); 1 week later right-sided RN neurons were axotomized by left-sided hemisection at C3-4 vertebral level, and Embryonic Day 14 spinal cord or gelfoam was introduced into the cavity. Additional rats received hemisection and a transplant of fetal spinal cord or gelfoam without FG injection. At 2 and 4 months, the number of neurons in the magnocellular portion of the RN contralateral to the hemisection decreased 35-40% in rats that received gelfoam; mean soma area of surviving neurons decreased 40%. RN cell loss was reduced to 20% in rats that received fetal spinal cord transplants, but the decrease in mean soma area was unchanged. Transplants therefore rescued about half of the axotomized RN neurons that otherwise would have died but did not prevent perikaryal atrophy. Anterograde transport of WGA-HRP injected into RN 2 months after transplantation showed that rubrospinal axons reached the site of injury but rarely entered transplants; FG injections caudal to transplants showed that axons of transplant neurons extended at least two segments into host spinal cord. Fetal spinal cord transplants may therefore contribute to locomotor recovery in adults with spinal cord injuries both by preventing retrograde cell death and by establishing novel circuits across the site of injury.

Grafts of fetal central nervous system tissue rescue axotomized Clarke's nucleus neurons in adult and neonatal operates.

Many conditions are thought to contribute to neuron death after axotomy, including immaturity of the cell at the time of injury, inability to reestablish or maintain target contact, and dependence on trophic factors produced by targets. Exogenous application of neurotrophic factors and transplants of peripheral nerve and embryonic central nervous system (CNS) tissue temporarily rescue axotomized CNS neurons, but permanent rescue may require transplants that are normal targets of the injured neurons. We examined the requirements for survival of axotomized Clarke's nucleus (CN) neurons. Two months after hemisection of the spinal cord at the T8 segment, there was an ipsilateral 30% loss of neurons at the L1 segment in adult operates and a 40% loss in neonates. Transplants of embryonic spinal cord, cerebellum, and neocortex inserted into the T8 segment at the time of hemisection prevented virtually all of the cell death in both adults and neonates, but transplants of embryonic striatum were ineffective. None of the grafts prevented the somal atrophy of CN neurons caused by axotomy. Retrograde transport of fluoro-gold from the cerebellum demonstrated that 33% of all CN neurons at L1 project to the cerebellum, 50% of these died following a T8 hemisection, but all these projection neurons were rescued by a transplant of embryonic spinal cord. These results suggest that the rescue of axotomized CN neurons is relatively specific for the normal target areas of these neurons, but this specificity is not absolute and may depend on the distribution and synthesis of particular neurotrophic agents.

Death of some dorsal root ganglion neurons and plasticity of others following sciatic nerve section in adult and neonatal rats.

Newborn animals recover from neurological injury to a greater extent than adults in spite of the greater vulnerability of developing neurons to retrograde or transneuronal degeneration (Kennard, '42; Goldman, '74; Prendergast and Stelzner, '76; Bregman and Goldberger, '82, '83). The cellular mechanisms underlying this "infant lesion effect" are incompletely understood (Bregman and Goldberger, '82). The dorsal root ganglion (DRG) is an excellent model in which to compare the developing and adult nervous system with respect to the effects of axotomy on cell survival and cellular function. We studied the survival of L5 DRG neurons after section-ligation of the sciatic nerve of adult and neonatal rats and used qualitative and quantitative immunocytochemical methods to examine changes in intraspinal substance P immunoreactivity (SPIR). Retrograde transport of wheatgerm agglutinin-horseradish (WGA-HRP) peroxidase applied to the sciatic nerve of adult or neonatal rats demonstrated that 70% of the neurons in the normal L5 DRG project into the sciatic nerve at the site of transection. In adults 20% of all L5 DRG neurons died between 10 and 60 days postoperative; in newborns 50% of the neurons died between 5 and 10 days. These results indicate that 30% of axotomized neurons in adults and 75% in neonates die after sciatic nerve section and that neuron loss is both more rapid and more extensive in neonates. No cell death was observed in the L5 DRG of neonates after dorsal rhizotomy, thus suggesting that at this stage of development the survival of DRG neurons depends on the peripheral but not the central process. SPIR in laminae I and II of both adult and newborn operates decreased and then recovered, but the time course and extent of the recovery differ. In adults SPIR was depleted in the medial portion of the L5 segment ipsilateral to surgery by 10 days postoperative and remained depleted for at least 2 months. By 1 year partial recovery occurred, but remained incomplete even at the longest survival time studied (15 months). SPIR, which is present in the dorsal horn at birth, was diminished in ipsilateral laminae I and II by 4 days after nerve section on the day of birth. Between 30 days and 60 days, the density of SPIR in the dorsal horn ipsilateral to surgery became virtually indistinguishable from that on the contralateral, intact side, suggesting a more rapid and complete recovery than in adults.(ABSTRACT TRUNCATED AT 400 WORDS)

Differentiation of substantia gelatinosa-like regions in intraspinal and intracerebral transplants of embryonic spinal cord tissue in the rat.

The differentiation of intracerebral and intraspinal transplants of fetal (E14-E15) rat spinal cord was studied to determine the extent to which myelin-free zones in these embryonic grafts exhibit cytological features and immunocytochemical characteristics of the substantia gelatinosa (SG) of the normal spinal cord. Immunocytochemical staining with antiserum to myelin basic protein (MBP) revealed myelin-free areas of varying proportions within fetal spinal cord grafts. These regions were identified in both newborn and adult recipients regardless of whether donor tissue was grafted to heterotopic (intracerebral) or homotopic (intraspinal) sites. As in the SG of the intact spinal cord, the myelin-free regions consisted mainly of small (7-15 microns) diameter neurons. At the ultrastructural level, these cells were surrounded by a neuropil composed of numerous small caliber, unmyelinated axons and intermediate-sized dendrites. Synaptic terminals in these areas were primarily characterized by the presence of clear, round vesicles, although granular vesicles were occasionally found within these terminals. Immunocytochemical staining demonstrated met- and leu-enkephalin-, neurotensin-, substance P-, and somatostatin-like immunoreactive elements within these myelin-free areas. Thus, regions within embryonic spinal cord grafts undergo some topographical differentiation which parallels that of the normal superficial dorsal horn. The presence of SG-like regions illustrates the potential capacity of fetal spinal cord transplants for replacing some intraspinal neuronal populations at the site of a spinal cord injury in neonatal and adult animals. These graft regions may serve as a source of intersegmental projection neurons or establish an extensive intrinsic circuitry similar to that seen in the normal SG. In addition, the definition of these areas provides a useful model to study the innervation patterns of host axons that typically project to the substantia gelatinosa of the normal spinal cord.

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.

Characterization of forms of immunoreactive somatostatin in sensory neuron and normal and deafferented spinal cord.

In order to determine the contribution made by primary sensory afferents and supraspinal projections to the immunoreactive somatostatin (IRS) content of the spinal cord, measurements were made of the concentration of IRS in the dorsal and ventral halves of the cord in cats subjected to unilateral lumbosacral dorsal rhizotomy (L1-S3) alone or combined with spinal cord transection. The molecular forms of IRS (characterized by gel chromatography) in L7 lumbar spinal cord, L6-S1 dorsal roots, ventral roots and dorsal root ganglia, and sciatic nerve were also determined. S14 was the predominant form in all tissues examined, but two additional molecular forms corresponding to S28 and S11.5 kdalton were present in dorsal root ganglia and spinal cord; S28 but not S11.5 kdalton was detected in both dorsal roots and sciatic nerves. These results indicate that S14 and S28 are transported along the central and peripheral processes of dorsal root ganglia, but that spinal cord S11.5 kdalton originates in the central nervous system. IRS in the dorsal horn was reduced by ca. 40% following dorsal root section. Neither disruption of descending pathways by spinal transection nor surgical isolation of the lumbar segments lowered cord somatostatin content below that produced by dorsal root section, indicating that most of the somatostatin within the cord arises from the dorsal root and from neurons in local spinal segments. Although the total content of IRS in the dorsal horn was reduced by ca. 40% following dorsal rhizotomy, the pattern of molecular forms was not changed accordingly.(ABSTRACT TRUNCATED AT 250 WORDS)

Sciatic nerve transection produces death of dorsal root ganglion cells and reversible loss of substance P in spinal cord.

Sciatic nerve section has been shown to reduce substance P (SP) in the dorsal horn of the spinal cord, but the mechanism which underlies the reduction is not understood. Whether SP levels subsequently recover as they do after dorsal rhizotomy has also been unknown. To test the hypothesis that transganglionic degeneration of primary afferents contributes to the reduction of SP, we have studied the changes in SP which result from section of the cat sciatic nerve and determined the extent of dorsal root ganglion (DRG) cell death. Sciatic nerve section resulted in DRG cell death, but the amount was variable and not seen in all animals. Reduction in dorsal horn and DRG SP was seen in all animals, and in the spinal cord it was followed by recovery. These sequelae resemble the changes which follow dorsal rhizotomy. After sciatic nerve section, the reduction in dorsal horn SP is smaller than after rhizotomy, the recovery more complete, and both the reduction and the recovery proceed more slowly. Evidence is presented that similar mechanisms may contribute to depletion of intraspinal SP after sciatic nerve section and after dorsal rhizotomy. The mechanisms contributing to recovery of spinal cord SP after sciatic nerve section may resemble known mechanisms of recovery that occur when the lesion is central.

Recovery of substance P but not somatostatin in the cat spinal cord after unilateral lumbosacral dorsal rhizotomy: a quantitative study.

The dorsal horn of the cat spinal cord contains substance P and somatostatin within nerve endings which arise from cells located in dorsal root ganglia and from cells within the neuraxis. Previous studies from this laboratory have demonstrated that dorsal rhizotomy depletes both peptides from the dorsal horn. However, the changes in the two peptides differ. Substance P is at first severely depleted by dorsal rhizotomy and then recovers in part, whereas somatostatin is diminished less but does not recover. In the present experiments the validity of these conclusions which were based on anatomical observations has been evaluated quantitatively with the use of radioimmunoassay. After a 74% reduction at 10-14 days postoperative, substance P immunoreactivity in the deafferented dorsal horn shows a small, statistically significant recovery by 30 days to 60% of normal values. In contrast, somatostatin is reduced by 46% at 10-14 days but does not return significantly. As previously suggested by immunocytochemistry, dorsal rhizotomy produces no significant decline of either peptide in the ventral horn. The differing response of the two peptides is consistent with the hypothesis that intrinsic spinal substance P-containing neurons increase their projections (or their production of substance P) in the deafferented dorsal horn, but that somatostatin-containing neurons do not. Because synaptic number returns to normal in at least the deafferented lamina II of the cat yet substance P recovers only partially, it is likely that axons which contain transmitters other than substance P or somatostatin also increase the numbers of their terminals in response to dorsal rhizotomy.