Development of serotonin immunoreactivity in the rat spinal cord and its plasticity after neonatal spinal cord lesions

The postnatal maturation of spinal pathways may account for the gradual time course of postnatal development of behavior and also account for the greater anatomical reorganization which often follows damage to the developing CNS compared to the mature CNS. The purpose of the current study was to examine (1) the prenatal and postnatal development of the descending serotonergic (5-HT) projection to the spinal cord and (2) the effects of a neonatal spinal cord lesion on this development. In addition, we wished to determine (3) whether transplants of fetal spinal cord tissue placed into the neonatal lesion site alter the plasticity of the 5-HT projection to the cord. Peroxidase-antiperoxidase immunocytochemical techniques were used. At embryonic day 14 (E14), no 5-HT immunoreactive fibers could be identified at any spinal cord level. By E18 the first axons were identified in the white matter only at all spinal cord levels. At birth, 5-HT immunoreactive fibers were present both in the white matter and in the gray matter at all cord levels. The projection within the gray matter was diffuse and considerably less dense than in the adult. The postnatal maturation of the 5-HT projection within the gray matter of the spinal cord followed rostral to caudal and ventral to dorsal gradients. During the first weeks postnatal, the 5-HT immunoreactivity within the cord increased to attain an adult pattern and density by 14 days in the cervical cord and 21 days in the thoracic and lumbar cord. The effect of a spinal cord hemisection at birth on the anatomical reorganization of the descending serotonergic innervation of the cord was compared with the effect of the same lesion in the adult. In the adult animal, mid-thoracic hemisection decreased the 5-HT content of the ventral horn of the lumbar spinal cord caudal and ipsilateral to the lesion to 8% of that on the intact side. When this same lesion was made in the newborn animal, the innervation was 43% of that on the intact side. When a transplant of fetal spinal cord tissue was inserted into the lesion site in the newborn animals, there was even greater 5-HT innervation caudal to the lesion, 83% of that on the intact side. These results indicate that there is considerable postnatal development and plasticity of the descending serotonergic projection to the spinal cord, and this plasticity is enhanced by the presence of a spinal cord transplant at the site of the lesion.     

A preliminary study of homotopic fetal cortical and spinal cotransplants in adult rats

Neocortical and spinal tissue from a given E16-17 rat fetus were homotopically transplanted into lesion sites of adult rats which had undergone combined cortical and complete lower thoracic spinal cord lesions. Spinal cord transplants were placed either directly into the gap in host spinal cord or embedded in a collagen matrix. Animals were killed from 4 days to 8 months and tissues were processed for light microscopy. All cortical transplants survived and integrated with host brain. Many axons appeared to grow between the cortical transplant and subjacent host parenchyma. Only collagen-embedded spinal transplants survived. At 8 months, two animals underwent spinal cord transection and HRP implantation two vertebral segments rostral to the spinal cord transplantation site. Both animals revealed HRP-labeled neurons in the cortical transplants. It was concluded that 1) homotopically transplanted fetal cortical tissue can survive and may be capable of extending axons to midthoracic levels, and 2) a collagen matrix may enhance the survival of fetal tissues transplanted into a complete gap in host spinal cord.     

Neural tissue transplants rescue axotomized rubrospinal cells from retrograde death

Rubrospinal tract cells undergo massive retrograde degeneration following spinal cord damage in newborn rats (Prendergast and Stelzner, J. Comp. Neurol. 166:163-172, '76b). In the current study, fetal spinal cord tissue (E12-14) was grafted into midthoracic spinal cord lesions in newborn rats (less than 72 hours old) in order to determine whether such transplants could modify the response of the immature host central nervous system (CNS) to axotomy. These transplants grew, differentiated, and formed extensive areas of apposition with the recipient spinal cords. Counts of red nucleus (RN) neurons indicated a significant loss of RN neurons in animals with lesion alone, but a rescuing of most of these cells if a transplant was placed into the lesion site. In fact, the number of neurons in animals with lesions and transplants was not significantly different from control animals. Horseradish peroxidase injected 10-15 mm caudal to the transplant (at 1-12 months post-transplantation) labeled neurons within the transplant and RN neurons contralateral to the spinal cord lesions and transplant. In animals with spinal cord lesion but no transplant, only the unaxotomized RN was labeled. Thus, spinal cord transplants prevented the massive retrograde cell death of immature axotomized rubrospinal neurons. Some of these rescued neurons projected to the host spinal cord caudal to the transplant.     

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.     

Direct agonists for serotonin receptors enhance locomotor function in rats that received neural transplants after neonatal spinal transection

We analyzed whether acute treatment with serotonergic agonists would improve motor function in rats with transected spinal cords (spinal rats) and in rats that received transplants of fetal spinal cord into the transection site (transplant rats). Neonates received midthoracic spinal transections within 48 hr of birth; transplant rats received fetal (embryonic day 14) spinal cord grafts at the time of transection. At 3 weeks, rats began 1-2 months of training in treadmill locomotion. Rats in the transplant group developed better weight-supported stepping than spinal rats. Systemic administration of two directly acting agonists for serotonergic 5-HT(2) receptor subtypes, quipazine and (+/-)-1-[2, 5]-dimethoxy-4-iodophenyl-2-aminopropane), further increased weight-supported stepping in transplant rats. The improvement was dose-dependent and greatest in rats with poor to moderate baseline weight support. In contrast, indirectly acting serotonergic agonists, which block reuptake of 5-HT (sertraline) or release 5-HT and block its reuptake (D-fenfluramine), failed to enhance motor function. Neither direct nor indirect agonists significantly improved locomotion in spinal rats as a group, despite equivalent upregulation of 5-HT(2) receptors in the lumbar ventral horn of lesioned rats with and without transplants. The distribution of immunoreactive serotonergic fibers within and caudal to the transplant did not appear to correspond to restoration of motor function. Our results confirm our previous demonstration that transplants improve motor performance in spinal rats. Additional stimulation with agonists at subtypes of 5-HT receptors produces a beneficial interaction with transplants that further improves motor competence.     

Plasticity of tyrosine hydroxylase and serotonergic systems in the regenerating spinal cord of adult zebrafish

Monoaminergic innervation of the spinal cord has important modulatory functions for locomotion. Here we performed a quantitative study to determine the plastic changes of tyrosine hydroxylase-positive (TH1(+); mainly dopaminergic), and serotonergic (5-HT(+)) terminals and cells during successful spinal cord regeneration in adult zebrafish. TH1(+) innervation in the spinal cord is derived from the brain. After spinal cord transection, TH1(+) immunoreactivity is completely lost from the caudal spinal cord. Terminal varicosities increase in density rostral to the lesion site compared with unlesioned controls and are re-established in the caudal spinal cord at 6 weeks post lesion. Interestingly, axons mostly fail to re-innervate more caudal levels of the spinal cord even after prolonged survival times. However, densities of terminal varicosities correlate with recovery of swimming behavior, which is completely lost again after re-lesion of the spinal cord. Similar observations were made for terminals derived from descending 5-HT(+) axons from the brain. In addition, spinal 5-HT(+) neurons were newly generated after a lesion and transiently increased in number up to fivefold, which depended in part on hedgehog signaling. Overall, TH1(+) and 5-HT(+) innervation is massively altered in the successfully regenerated spinal cord of adult zebrafish. Despite these changes in TH and 5-HT systems, a remarkable recovery of swimming capability is achieved, suggesting significant plasticity of the adult spinal network during regeneration.     

Fetal cell grafts into resection and contusion/compression injuries of the rat and cat spinal cord.

This article reviews recent findings concerning the feasibility, basic neurobiology, and potential functional benefits of fetal CNS tissue grafts into acute and chronic lesions of the adult spinal cord. In the rat, neuro-anatomical observations suggest that transplants into resection cavities establish neuritic projections that could functionally reunite separated rostral and caudal segments of the host spinal cord. Furthermore, some complementary electrophysiological evidence has been obtained for synaptic connectivity between host and graft neurons. In these studies, extracellular single-unit activity was evoked in fetal spinal cord (FSC) transplants by stimulating host dorsal roots that had been juxtaposed to donor tissue at the time of transplantation. In other investigations, we examined whether grafts could also establish axonal projections to appropriate areas of gray matter in the chronically injured spinal cord. For this purpose, fetal serotoninergic (5-HT) neurons were injected caudal to complete spinal cord transections that had been made 1-3 months earlier. Immunocytochemistry revealed that these cells projected their axons into gray matter regions normally innervated by bulbospinal 5-HT neurons. To investigate transplantation in a more clinically relevant lesion model, a third group of experiments involved injection of dissociated cell suspensions into acute [less than 24 h postinjury (p.i.)]), subchronic (7-10 days p.i), and chronic (greater than or equal to one month, p.i.) contusion lesions. Such grafts routinely filled areas that otherwise would have been regions of cavitation extending rostral-caudal distances of approximately 7 mm. FSC transplants in such injuries also appeared to influence some aspects of motoneuron excitability and hindlimb locomotion. More recent studies of the cat spinal cord have extended these findings in the rat by showing long-term survival (greater than 2 years) of fetal CNS allografts in recipients with either subtotal transection or compression lesions. Preliminary studies of connectivity have also shown host-graft projection patterns similar to those seen in the rat. Behavioral analyses are currently underway to examine the effects of fetal grafts in cats with chronic postcompression lesions. These observations in the rat and cat are discussed in the general context of basic biological and clinical issues relevant to the long-term objective of promoting functional improvement in the damaged spinal cord.     

Intraspinal transplantation of embryonic spinal cord tissue in neonatal and adult rats

Fetal rat spinal cord tissue was obtained on gestational day 14 (E14) and transplanted into 2-4-mm-long intraspinal cavities produced by partial spinal cord lesions in adult and neonatal rats. At regular post-transplantation intervals, light and electron microscopy, autoradiographic demonstration of tritiated thymidine labelling, and immunocytochemical localization of glial fibrillary acidic protein (GFAP) were used to identify surviving donor tissues and to study their differentiation and extent of fusion with recipient spinal cords. In some experiments, wheat germ agglutinin-conjugated horseradish peroxidase (WGA-HRP) was also employed to examine whether neurons within the grafts projected axons into the host spinal cord and vice versa. Lastly, immunocytochemistry was used to determine whether any supraspinal serotoninergic (5-HT) axons from the host extended into the transplants. Over 80% of the grafts survived in lesions of both the neonatal and adult rat spinal cord for periods of 1-16 months (duration of experiment), and considerable maturation of donor tissue was evidenced, which even included the appearance of some topographical features of the normal spinal cord. Many of the transplants extended the entire length of the lesion, and were often closely apposed to the injured surfaces of the recipient spinal cords without an intervening dense glial scar. At post-transplantation intervals of 2-4 months, injection of WGA-HRP into the host spinal cord (5 mm from the transplant in adult animals or as much as 20 mm in neonatal recipients) demonstrated retrogradely labelled neurons and anterogradely labelled axons in the grafts. Likewise, injecting WGA-HRP into transplants in adult recipients resulted in labelling of neurons in adjacent segments of the host spinal cord; some labelled axons, derived from donor neurons, were also present in neighboring spinal gray matter. Finally, immunocytochemistry revealed 5-HT-like immunoreactive fibers in transplants that had been prelabelled with tritiated thymidine. These observations demonstrate the potential of embryonic spinal cord transplants to replace damaged intraspinal neuronal populations and to restore some degree of anatomical continuity between the isolated rostral and caudal stumps of the injured mammalian spinal cord.     

A re-assessment of the consequences of delayed transplantation of olfactory lamina propria following complete spinal cord transection in rats

This study is part of the NIH "Facilities of Research-Spinal Cord Injury" contract to support independent replication of published studies. We repeated a study reporting that delayed transplantation of olfactory lamina propria (OLP) into the site of a complete spinal cord transection led to significant improvement in hindlimb motor function and induced axon regeneration. Adult female rats received complete spinal cord transections at T10. Thirty days post-injury, pieces of OLP, which contains olfactory ensheathing cells (OECs), or respiratory lamina propria (RLP), which should not contain OECs, were placed into the transection site. Hindlimb motor function was tested using the BBB scale from day 1 post-injury through 10 weeks following transplantation. To assess axonal regeneration across the transection site, Fluorogold was injected into the distal segment, and the distribution of 5HT-containing axons was assessed using immunostaining. BBB analyses revealed no significant recovery after OLP transplantation and no significant differences between OLP vs. RLP transplant groups. Fluorogold injections into caudal segments did not lead to retrograde labeling in any animals. Immunostaining for 5HT revealed that a few 5HT-labeled axons extended into both RLP and OLP transplants and a few 5HT-labeled axons were present in sections caudal to the injury in 2 animals that received OLP transplants and 1 animal that received RLP transplants. Our results indicate that, although OLP transplants may stimulate regeneration under some circumstances, the effect is not so robust as to reliably overcome the hostile setting created by a complete transection paradigm.     

Both regenerating and late-developing pathways contribute to transplant-induced anatomical plasticity after spinal cord lesions at birth.

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)     

Neurotrophic Factors Increase Axonal Growth after Spinal Cord Injury and Transplantation in the Adult Rat

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 μm 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 aneurotropicinfluence on injured mature CNS neurons by increasing their axonal growth within a transplant.     

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.     

Evidence for growth of supraspinal axons through the lesion after transection of the thoracic spinal cord in the developing opossum Didelphis virginiana

In the present study, we asked whether supraspinal axons grow through a complete transection of the spinal cord in the developing opossum Didelphis virginiana. When the thoracic cord was transected at postnatal day (PD) 5 and bilateral injections of Fast Blue (FB) were made four segments caudal to the lesion 30–40 days later, FB-containing neurons were found in each of the supraspinal nuclei labeled by comparable injections in age-matched unlesioned controls. Continuity between the cut ends of the cord was obvious grossly when the animals were killed, and histologically recognizable spinal cord was present at the lesion site. When the same procedure was followed on pups subjected to transection at PD12, FB-containing neurons were still present at supraspinal levels, but they appeared to be fewer in number than in the PD5 cases or the age-matched controls, and none were found within the medial pontine reticular and lateral vestibular nuclei. When the lesion was made at PD20, labeled neurons were even fewer in number, and when it was made at PD26, they were restricted to the medullary raphe and the red nuclei. There was no evidence for growth of supraspinal axons across lesions made at PD33. We conclude that supraspinal axons grow through the lesion after transection of the spinal cord in neonatal opossums and that the critical period for growth of reticulospinal and vestibulospinal axons through the lesion ends earlier than that for comparable growth of raphespinal and rubrospinal axons. © 1996 Wiley-Liss, Inc.     

Ascending tract neurons survive spinal cord transection in the neonatal rat

Retrograde axonal transport was used to determine which ascending nerve tracts from the lumbosacral spinal cord are present in the cervical spinal cord of the newborn rat and if their cell bodies survive axotomy. A pledget of true blue was applied to a low cervical spinal transection in the newborn rat (N = 4). After a 5-day survival period, neurons were labeled in the laminae of origin of all ascending nerve tracts throughout the lumbosacral spinal cord. Neurons labeled in the same way survived for at least 1 month postoperatively when the spinal cord was transected at a midthoracic level at 5 days of age (N = 4). No neurons in the lumbosacral spinal cord were labeled if the midthoracic spinal cord was transected at the same time as application of the dye to cervical spinal cord (N = 2). Therefore, neurons labeled with true blue from cervical spinal cord during the neonatal period are likely to have been axotomized by thoracic injury made at 5 days of age. Three months after midthoracic spinal transection of newborn rats, HRP was injected or a pledget was applied to the first spinal segment caudal to this lesion (N = 8). The same population of neurons was labeled as in adult rats receiving application of HRP to an acute midthoracic spinal transection (N = 4). Neurons were seldom labeled in adult rats in which HRP was injected and ascending nerve tract axons not damaged (N = 4). These results suggest that most ascending nerve tract axons are present in cervical spinal cord during the neonatal period (by 4 to 5 days of age).(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Axonal projections between fetal spinal cord transplants and the adult rat spinal cord: a neuroanatomical tracing study of local interactions.

Three neuroanatomical tracers have been employed to map the axonal projections formed between transplants of fetal spinal cord tissue and the surrounding host spinal cord in adult rats. Solid pieces of embryonic day 14 (E14) rat spinal cord were placed into hemisection aspiration cavities in the lumbar spinal cord. Injections of either (1) a mixture of horseradish peroxidase and wheat germ agglutinin- conjugated horseradish peroxidase, (2) Fluoro-Gold, or (3) Phaseolus vulgaris leucoagglutinin (PHA-L) were made into the transplants or the neighboring segments of the host spinal cord at 6 weeks to 14 months post-transplantation. Injections of anterograde and retrograde tracers into the transplants revealed extensive intrinsic projections that often spanned the length of the grafts. Axons arising from the transplants extended into the host spinal cord as far as 5 mm from the host-graft interface, as best revealed by retrograde labeling with Fluoro-Gold. Consistent with these observations, iontophoretic injections of PHA-L into the transplants also produced labeled axonal profiles at comparable distances in the host spinal cord, and in some instances elaborate terminals fields were observed surrounding host neurons. The majority of these efferent fibers labeled with PHA-L, however, were confined to the immediate vicinity of the host-graft boundary, and no fibers were seen traversing cellular partitions between host and transplant tissues. Host afferents to the transplants were also revealed by these tracing methods. For example, the injection of Fluoro-Gold into the grafts resulted in labeling of host neurons within the spinal cord and nearby dorsal root ganglia. In most cases, retrogradely labeled neurons in spinal gray matter were located within 0.5 mm of the graft site, although some were seen as far as 4-6 mm away. The distance and relative density of ingrowth exhibited by host axons into the grafts, however, appeared modest based upon the results of HRP and Fluoro-Gold retrograde labeling. This was further confirmed with the PHA-L anterograde method. Whereas some host fibers were seen extending into the transplants, the majority of PHA-L containing axons formed terminal-like profiles at or within 0.5 mm of the host-graft interface. The comprehensive view of intrinsic connectivity and host-graft projections obtained in these studies indicates that intraspinal grafts of fetal spinal cord tissue can establish a short-range intersegmental circuitry in the injured, adult spinal cord. These observations are consistent with the view that such grafts may contribute to the formation of a functional relay between separated segments of the spinal cord after injury.(ABSTRACT TRUNCATED AT 400 WORDS)     

Transplanting neural progenitors into a complete transection model of spinal cord injury

Neural progenitor cell (NPC) transplantation is a promising therapeutic strategy for spinal cord injury (SCI) because of the potential for cell replacement and restoration of connectivity. Our previous studies have shown that transplants of NPC, composed of neuron‐ and glia‐restricted progenitors derived from the embryonic spinal cord, survived well in partial lesion models and generated graft‐derived neurons, which could be used to form a functional relay. We have now examined the properties of a similar NPC transplant using a complete transection model in juvenile and adult rats. We found poor survival of grafted cells despite using a variety of lesion methods, matrices, and delays of transplantation. If, instead of cultured progenitor cells, the transplants were composed of segmental or dissociated segments of fetal spinal cord (FSC) derived from similar‐staged embryos, grafted cells survived and integrated well with host tissue in juvenile and adult rats. FSC transplants differentiated into neurons and glial cells, including astrocytes and oligodendrocytes. Graft‐derived neurons expressed glutaminergic and GABAergic markers. Grafted cells also migrated and extended processes into host tissue. Analysis of axon growth from the host spinal cord showed serotonin‐positive fibers and biotinylated dextran amine‐traced propriospinal axons growing into the transplants. These results suggest that in treating severe SCI, such as complete transection, NPC grafting faces major challenges related to cell survival and formation of a functional relay. Lessons learned from the efficacy of FSC transplants could be used to develop a therapeutic strategy based on neural progenitor cells for severe SCI. © 2014 Wiley Periodicals, Inc.     

移植嗅鞘细胞可通过减弱纤维性瘢痕的产生而促进大鼠脊髓损伤后神经纤维的再生

背景：多项研究已证实嗅鞘细胞移植能促进脊髓损伤大鼠神经再生和功能的恢复,但嗅鞘细胞移植促进再生的分子机制还未完全阐明。 目的：观察嗅鞘细胞移植是否可以消除纤维性瘢痕的产生。 设计、时间及地点：对照随机动物实验,于2007年4月至2009年5月在日本东京都神经科学研究所发生形态部门和中国医科大学基础医学院解剖教研室完成。 材料：动物由日本东京都神经科学研究所动物管理及使用委员会提供 方法：选取体重为300-350克的SD大鼠23只,行胸椎9-10脊髓全横断,分为假手术对照组（n=3）、手术组（n=9）和嗅鞘细胞移植组（n=11）。嗅鞘细胞来源于未成熟的嗅球,并经过培养2-3周。 主要观察指标：分别应用GFAP和胶原IV蛋白免疫组化染色观察损伤部位胶质瘢痕和纤维性瘢痕的形成。脊髓内下行和上行的神经纤维束分别应用5-羟色胺和降钙素基因相关肽组化染色来观察。血管可用RECA-1染色来标识。同一切片上用RECA-1和胶原IV染色来比较血管的着色和胶原沉着的部位。 结果：在单纯的脊髓损伤组,损伤后一周的下行的5-羟色胺阳性纤维和上行的降钙素基因相关肽神经纤维均停止在纤维性瘢痕两端,在损伤部位周边有大量胶质细胞增生,损伤中心部位有大量胶原IV蛋白沉积而形成纤维性瘢痕。在损伤后同时移植嗅鞘细胞一周,5-羟色胺和降钙素基因相关肽阳性纤维均可见通过损伤部位。在损伤的脊髓节段,嗅鞘细胞移植组的阳性纤维均高于单纯损伤组。虽然移植组的损伤周边仍有大量的胶质细胞存在,但纤维性瘢痕却被显著的抑制。 结论：脊髓损伤过程中切断的神经纤维停止在纤维性瘢痕之前,移植嗅鞘细胞可以减弱纤维性瘢痕的产生而促进5-羟色胺阳性纤维越过脊髓损伤部位。     

Regeneration of descending spinal axons after transection of the thoracic spinal cord during early development in the North American opossum, Didelphis virginiana

Opossums are born in an immature, fetal-like state, making it possible to lesion their spinal cord early in development without intrauterine surgery. When the thoracic spinal cord of the North American opossum, Didelphis virginiana, is transected on postnatal day 5, and injections of Fast Blue (FB) are made caudal to the lesion site 30-40 days or 6 months later, neurons are labeled in all of the spinal and supraspinal areas that are labeled after comparable injections in age-matched, unlesioned controls. Double-labeling studies document that regeneration of cut axons contributes to growth of axons through the lesion site and behavioral studies show that animals lesioned on postnatal day 5 use their hindlimbs in normal appearing locomotion as adults. The critical period for developmental plasticity of descending spinal axons extends to postnatal day 26, although axons which grow through the lesion site become fewer in number and more restricted as to origin with increasing age. Animals lesioned between postnatal day 12 and 26 use the hindlimbs better than animals lesioned as adults, but hindlimb function is markedly abnormal and uncoordinated with that of the forelimbs. We conclude that restoration of anatomical continuity occurs after transection of the spinal cord in developing opossums, that descending axons grow through the lesion site, that regeneration of cut axons contributes to such growth, and that animals lesioned early enough in development have relatively normal motor function as adults.     

Induced expression of polysialic acid in the spinal cord promotes regeneration of sensory axons

After spinal cord injury axonal regeneration is prevented by glial scar formation. In this study we examined whether induced expression of polysialic acid (PSA) in the lesion site would render the glial scar permissive to axonal regeneration after dorsal column transection. PSA was induced by lentiviral vector-mediated expression of polysialyltransferase (LV/PST). PSA expression increased astrocyte infiltration and permitted the penetration of regenerating axons across the caudal border of the lesion and into the lesion cavity. In LV/PST-injected animals with a peripheral nerve-conditioning lesion, 20 times more axons grew into the lesion cavity than those LV/GFP-injected plus conditioning lesion, and some axons grew across the cavity and extended to the rostral cord, while in LV/GFP group most ascending axons terminated at the caudal border of the lesion. Our result suggests that induced expression of PSA can provide a favorable environment for axonal regeneration.