Transplantation of olfactory ensheathing cells promotes axonal regeneration in a rat model of spinal cord injury

BACKGROUND:Transplantation of olfactory ensheathing cells (OECs) into the injured spinal cord has been shown to promote axonal regeneration and functional recovery.However,the mechanisms underlying the effects of OEC transplantation remain controversial.OBJECTIVE:To observe fibrotic scar formation and axonal regeneration in the damaged spinal cord following OEC transplantation,and to determine whether OEC transplantation promotes neural regeneration by attenuating fibrotic scar formation.DESIGN,TIME AND SETTING:A randomized,controlled animal experiment was performed at the Department of Developmental Morphology,Tokyo Metropolitan Institute for Neuroscience,Fuchu,Japan and at the Department of Human Anatomy,College of Basic Medical Sciences,China Medical University,China between April 2007 and May 2009.MATERIALS:OECs were obtained from olfactory nerves and olfactory bulbs of male,4-week-old,Sprague Dawley rats.Rabbit anti-serotonin polyclonal antibody,rabbit anti-calcitonin gene-related peptide polyclonal antibody,rabbit anti-glial fibrillary acidic protein polyclonal antibody,rabbit anti-type IV collagen polyclonal antibody,and mouse anti-rat endothelial cell antigen-1 monoclonal antibody were used.METHODS:Male,Sprague Dawley rats aged 8 weeks were randomly divided into three groups:sham-surgery (n = 3),surgery (n = 9),and OEC transplantation (n = 11).Spinal cord transection at the T9-10 level was performed and the rats were transplanted with a 2-μL (1 × 105 cells) cell suspension.MAIN OUTCOME MEASURES:Formation of glial and fibrotic scars was examined using immunohistochemistry for glial fibrillary acidic protein and type IV collagen.Serotonin-positive and calcitonin gene-related peptide-positive axons were visualized by immunohistochemistry,respectively.Double immunofluorescence for type IV collagen and rat endothelial cell antigen-1 was also performed to determine co-localization of type IV collagen deposition and blood vessels.RESULTS:At 1 week after spinal cord injury,numerous glial cells were observed around the lesion site.Formation of fibrotic scar was determined by a large amount of type IV collagen deposition in the lesion center,and descending serotonin- or ascending calcitonin gene-related peptideconiaining axons stopped at the fibrotic scar that was formed in the lesion site.At week after transplantation,the formation of fibrotic scar was significantly inhibited.In addition,the fibrotic structure was partly formed and centralized in the blood vessel,and serotonergic and calcitonin gene-related peptide-containing axons were regenerated across the lesion site.CONCLUSION:OEC transplantation into the injured spinal cord attenuated fibrotic scar formation and promoted axon regeneration.     

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.     

Regeneration of supraspinal axons after complete transection of the thoracic spinal cord in neonatal opossums (Monodelphis domestica)

These studies define the time table and origin of supraspinal axons regenerating across a complete spinal transection in postnatal Monodelphis domestica. After lumbar (L1) spinal cord injection of fluorophore-dextran amine conjugate on postnatal (P) day 4, a consistent number of neurons could be labeled. The numbers of labeled neurons remained stable for several weeks, but subsequently declined by P60 in control animals and by P35 in animals with complete spinal transection (T4-T6) performed at P7. In control animals, 25-40% of neurons labeled with a fluorophore injected (L1) at P4 could also be double-labeled by a second fluorophore injected (T8-T10) at different older ages. In spinally transected animals, total numbers of neurons labeled with the second marker were initially lower compared with age-matched controls, but were not significantly different by 3 weeks after injury. The proportion of double-labeled neurons in spinally transected animals increased from approximately 2% 1 week after injury (P14) to approximately 50% by P60, indicating that a substantial proportion of neurons with axons transected at P7 is able to regenerate and persist into adulthood. However, the proportion of axons originating from regenerating neurons made only a small contribution at older ages to total numbers of fibers growing through the injury site, because much of development of the spinal cord occurs after P7. Evidence was obtained that degenerating neurons with both apoptotic and necrotic morphologies were present in brainstem nuclei; the number of neurons with necrotic morphology was much greater in the brainstem of animals with spinal cords transected at P7.     

Spinal cord repair in neonatal rats: a correlation between axonal regeneration and functional recovery

The present study aimed to analyse how anatomical regeneration contributes to functional recovery after experimental spinal cord repair. Thoracic spinal cord of neonatal rats was completely transected to make a gap and repaired by grafting a section of embryonic spinal cord. Six weeks after surgery, outcome of locomotor performance was assessed using an open field locomotor scale (BBB scale). Axonal regeneration across the repaired site was quantitatively assessed in the raphe, vestibular, and red nuclei and the sensorimotor cortex by a retrograde tracing method. The rats that had no labelled neurons in any of the supraspinal nuclei showed no hind-forelimb coordination. The rats that had labelled neurons in the brainstem nuclei but not in the sensorimotor cortex showed hind-forelimb coordination of varying grades depending on the amount of regeneration. The rats that had labelled neurons in all of the examined nuclei showed almost normal locomotion. In addition to a relationship between distribution of the labelled neurons and functional recovery, a positive correlation was observed between number of the labelled neurons in each of the supraspinal nuclei and locomotor performance of the rat. Thus the grade of restored function appeared to be regulated by distribution and number of fibres regenerated across the repaired site and into the target region. These results suggest that accurate reconstruction of neural connections is essential for significant functional recovery after spinal cord repair.     

Adult neurogenesis in primate and rodent spinal cord: comparing a cervical dorsal rhizotomy with a dorsal column transection

Neurogenesis has not been shown in the primate spinal cord and the conditions for its induction following spinal injury are not known. In the first part of this study, we report neurogenesis in the cervical spinal dorsal horn in adult monkeys 6-8 weeks after receiving a well-defined cervical dorsal rhizotomy (DRL). 5-bromo-2-deoxyuridine (BrdU) was administered 2-4 weeks following the lesion. Cells colabeled with BrdU and five different neuronal markers were observed in the peri-lesion dorsal horn 4-5 weeks after BrdU injection. Those colabeled with BrdU and neuron-specific nuclear protein, and BrdU and glial fibrillary acidic protein were quantified in the dorsal horn peri-lesion region, and the ipsi- and contralateral sides were compared. A significantly greater number of BrdU/neuron-specific nuclear protein- and BrdU/glial fibrillary acidic protein-colabeled cells were found on the lesion side (P<0.01). These findings led us to hypothesize that neurogenesis can occur within the spinal cord following injury, when the injury does not involve direct trauma to the cord and glial scar formation. This was tested in rats. Neurogenesis and astrocytic proliferation were compared between animals receiving a DRL and those receiving a dorsal column lesion. In DRL rats, neurogenesis was observed in the peri-lesion dorsal horn. In dorsal column lesion rats, no neurogenesis was observed but astrocytic activation was intense. The rat data support our hypothesis and findings in the monkey, and show that the response is not primate specific. The possibility that new neurons contribute to recovery following DRL now needs further investigation.     

BD PuraMatrix peptide hydrogel as a culture system for human fetal Schwann cells in spinal cord regeneration

BD PuraMatrix peptide hydrogel, a three‐dimensional cell culture model of nanofiber scaffold derived from the self‐assembling peptide RADA16, has been applied to regenerative tissue repair in order to develop novel nanomedicine systems. In this study with PuraMatrix, self‐assembling nanofiber scaffold (SAPNS) and Schwann cells (SCs) were isolated from human fetal sciatic nerves, cultured within SAPNS, and then transplanted into the spinal cord after injury (SCI) in rats. First, the peptide nanofiber scaffold was evaluated via scanning electron microscopy and atomic force microscopy. With phase‐contrast microscopy, the appearance of representative human fetal SCs encapsulated in PuraMatrix on days 3, 5, and 7 in 12‐well plates was revealed. The Schwann cells in PuraMatrix were cultured for 2 days, and the SCs had active proliferative potential. Spinal cord injury was induced by placing a 35‐g weight on the dura of T9–T10 segments for 15 min, followed by in vivo treatment with SAPNS and human fetal SCs (100,000 cells/10 μl/injection) grafted into spinal cord 7 days after SCI. After treatment, the recovery of motor function was assessed periodically using the Basso, Beattie, and Bresnahan scoring system. Eight weeks after grafting, animals were perfusion fixed, and the survival of implanted cells was analyzed with antibody recognizing SCs. Immunohistochemical analysis of grafted lumber segments at 8 weeks after grafting revealed reduced asterogliosis and considerably increased infiltration of endogenous S100⁺ cells into the injury site, suggesting that PuraMatrix may play an important role in the repair observed after SAPNS and human fetal SC transplantation. © 2012 Wiley Periodicals, Inc.     

Tenascin-C expression and axonal sprouting following injury to the spinal dorsal columns in the adult rat

We have examined the expression and distribution of the extracellular matrix molecule tenascin-C in and around lesions of the thoracic dorsal columns in adult rats 3 days to 8 weeks after injury, using in situ hybridization, immunofluorescence, electron microscopy and immunoelectron microscopy. Numerous tenascin-C mRNA+ cells were present in and around the lesion at 3 days; fewer were present at 14 days and almost none 30 days after injury. Most tenascin-C mRNA+ cells in the spinal cord around the lesion were GFAP+, but most of those within the lesion were not, suggesting that tenascin-C is produced in the injured spinal cord by a subpopulation of astrocytes and by other cells that invade the lesion; these cells may include meningeal cells, macrophages, and Schwann cells. From 3 to 30 days after injury, heavy tenascin-C immunoreactivity was present at the lesion site (especially transections), and there was lighter immunoreactivity around the lesion and in the degenerating dorsal column. The heaviest immunoreactivity was associated with collagen fibrils in areas of expanded extracellular space and with basal laminae (covering Schwann cells and some astrocytes) but tenascin-C was also found close to the surfaces of some OX-42+ macrophages/microglia, leptomeningeal cells, and capillaries. Neurofilament (NF)+ axons grew into the highly tenascin-C-immunoreactive lesion sites, indicating that tenascin-C does not prevent axonal growth into these areas. However, such axons were not coated with tenascin-C except where directly exposed to the extracellular space. J. Neurosci. Res. 49:433–450, 1997. © 1997 Wiley-Liss, Inc.     

Localization of last-order premotor interneurons in the lumbar spinal cord of rats

There is strong evidence that neural circuits underlying certain rhythmic motor behaviors are located in the spinal cord. Such local central pattern generators are thought to coordinate the activity of motoneurons through specific sets of last-order premotor interneurons that establish monosynaptic contacts with motoneurons. After injections of biotinylated dextran amine into the lateral and medial motor columns as well as the ventrolateral white matter at the level of the upper and lower segments of the lumbar spinal cord, we intended to identify and localize retrogradely labelled spinal interneurons that can likely be regarded as last-order premotor interneurons in rats. Regardless of the location of the injection site, labelled interneurons were revealed in laminae V–VIII along a three- or four-segment-long section of the spinal gray matter. Although most of the stained cells were confined to laminae V–VIII in all cases, the distribution of neurons within the confines of this area varied according to the site of injection. After injections into the lateral motor column at the level of the L4–L5 segments, the labelled neurons were located almost exclusively in laminae V–VII ipsilateral to the injection site, and the perikarya were distributed throughout the entire mediolateral extent of this area. Interneurons projecting to the lateral motor column at the level of the L1–L2 segments were also located in laminae V–VII, but most of them were concentrated in the middle one-third or in the lateral half of this area. Following injections into the medial motor column at the level of the L1–L2 segments, the majority of labelled neurons were confined to the medial aspect of laminae V–VII and lamina VIII, and the proportion of neurons that were found contralateral to the injection site was strikingly higher than in the other experimental groups. The results suggest that the organization of last-order premotor interneurons projecting to motoneurons, which are located at different areas of the lateral and medial motor columns and innervate different muscle groups, may present distinct features in the rat spinal cord. J. Comp. Neurol. 389:377–389, 1997. © 1997 Wiley-Liss, Inc.     

Somatostatinergic neurons in the insular cortex project to the spinal cord: combined retrograde axonal transport and immunohistochemical study

A double-labeling method combining immunohistochemistry and a retrograde tracer technique using biotin-horseradish peroxidase (B-HRP) was employed to identify a descending somatostatinergic fiber system from the insular cortex to the spinal cord. Injection of B-HRP into the spinal cord at cervical or lumbar levels resulted in the labeling of a number of neurons in the insular cortex. Simultaneous immunostaining revealed the existence of double-labeled neurons in the insular cortex. The result provides direct evidence for the presence of a descending somatostatinergic pathway from the insular cortex to lumbar levels of the spinal cord.     

Glial and axonal responses in areas of Wallerian degeneration of the corticospinal and dorsal ascending tracts after spinal cord dorsal funiculotomy

Wallerian degeneration (WD), composed of the breakdown and phagocytosis of damaged axons and their myelin sheaths distal to the injury, is a major sequela of spinal cord injury (SCI). To understand the microenvironment within WD that may affect repair following SCI, we investigated the fate of major glial types and axons in this region following acute (1 h), subacute (10 days), and chronic (30 days) dorsal funiculotomy at the eighth thoracic (T8) level. This lesion induces a confined WD in two distinct functional pathways, that is, the corticospinal tract (CST) and dorsal ascending tract (DAT) in opposite directions. Here we report that astrocytes, reactive microglia and macrophages were all significantly increased in areas of WD in both the CST and DAT at subacute and chronic stages compared to the sham‐operated or acute stage. While the level of GFAP⁺ astrocytes remained stable after the subacute stage, the number of OX‐42⁺ microglia and ED‐1⁺ macrophages markedly decreased at the chronic stage. Interestingly, a mild but significant increase in ED‐1⁺ macrophages was also found in the intact fiber tracts 3 mm proximal to the injury at the chronic stage, coinciding with axonal dieback observed at that level. Axons distal to the injury experienced a continued and prolonged degeneration in both fiber tracts. Finally, although a significant decrease of Olig2⁺ oligodendrocyte lineage (OL) cells was found in areas of WD, the presence of these cells at the chronic stage indicates that they are available for endogenous repair. Taken together, our data have provided spatiotemporal evidence for the dynamic pathogenic changes of major cellular components in areas of WD remote to an SCI. Information obtained in this study should be useful for designing experiments aimed at modifying this region to accommodate endogenous or exogenous repair following SCI.     

Support of axonal regrowth by endogenous mechanisms following spinal cord injury in adult rats

Because of its non‐invasive nature and ease of regulation, a closely monitored cryogenic method of tissue injury was used to create a degree of spinal cord injury within which there would be an extended regrowth of axons. The parameters of cooling used in the present study resulted in an injury length of 1 cm through which 3 mm of measured axonal regrowth and 8 mm of observed regrowth occurred over a 56‐day period in the ascending fibers of the dorsal column of the mature rat. This was associated with the development of a cellular matrix consisting of macrophages, macroglia and Schwann cells which gradually expands within the injured area initially dominated by macrophages. It is the authors' impression that the presence of a substantial microglial component within the macrophage population may be a significant factor in the success of the axonal regrowth. Under this influence and that of the invading axons, the astrocyte, which provides the immediate cell support to the growing axon, can be maintained in a functional state that is supportive and not obstructive to the axon, presumably through the recruitment of astrocyte precursors from an indigenous stem cell population. These tissue changes indicate that adult mammalian spinal cord tissue does have the capacity to develop on its own a matrix capable of supporting the regrowth of axons.     

GDNF‐enhanced axonal regeneration and myelination following spinal cord injury is mediated by primary effects on neurons

We previously demonstrated that coadministration of glial cell line‐derived neurotrophic factor (GDNF) with grafts of Schwann cells (SCs) enhanced axonal regeneration and remyelination following spinal cord injury (SCI). However, the cellular target through which GDNF mediates such actions was unclear. Here, we report that GDNF enhanced both the number and caliber of regenerated axons in vivo and increased neurite outgrowth of dorsal root ganglion neurons (DRGN) in vitro, suggesting that GDNF has a direct effect on neurons. In SC‐DRGN coculture, GDNF significantly increased the number of myelin sheaths produced by SCs. GDNF treatment had no effect on the proliferation of isolated SCs but enhanced the proliferation of SCs already in contact with axons. GDNF increased the expression of the 140 kDa neural cell adhesion molecule (NCAM) in isolated SCs but not their expression of the adhesion molecule L1 or the secretion of the neurotrophins NGF, NT3, or BDNF. Overall, these results support the hypothesis that GDNF‐enhanced axonal regeneration and SC myelination is mediated mainly through a direct effect of GDNF on neurons. They also suggest that the combination of GDNF administration and SC transplantation may represent an effective strategy to promote axonal regeneration and myelin formation after injury in the spinal cord. © 2009 Wiley‐Liss, Inc.     

Spontaneous longitudinally orientated axonal regeneration is associated with the Schwann cell framework within the lesion site following spinal cord compression injury of the rat

Spontaneous cellular reorganisation at the lesion site has been investigated following massive spinal cord compression injury in adult rats. By 2 days post operation (p.o.), haemorrhagic necrosis, widespread axonal degeneration, and infiltration by polymorphnuclear granulocytes and OX42-positive macrophages were observed in the lesion site. By 7 days p.o., low affinity nerve growth factor receptor-positive Schwann cells, from activated spinal roots, were identified as they migrated far into the lesion. Between 7 and 14 days p.o., the overlapping processes of Schwann cells within the macrophage-filled lesion formed a glial framework which was associated with extensive longitudinally orientated ingrowth by many neurofilament-positive axons. Relatively few of these axons were calcitonin gene-related peptide (CGRP)-, substance P (SP)-, or serotonin (5HT)-positive; however, many were glycinergic or gamma aminobutyric acid (GABA)ergic. At 21 and 28 days p.o. (the longest survival times studied), a reduced but still substantial amount of orientated Schwann cells and axons could be detected at distances of up to 5 mm within the lesion. Glial fibrillary acidic protein (GFAP) immunoreactivity demonstrated the slow formation of astrocytic scarring which only became apparent at the lesion interface between 21 and 28 days p.o. The current data suggest the possibility of developing future therapeutic strategies designed to maintain or even enhance these spontaneous and orientated regenerative events. J. Neurosci. Res. 53:51–65, 1998. © 1998 Wiley-Liss, Inc.     

Major vault protein promotes locomotor recovery and regeneration after spinal cord injury in adult zebrafish

In contrast to mammals, adult zebrafish recover locomotor functions after spinal cord injury (SCI), in part due to axonal regrowth and regeneration permissivity of the central nervous system. Upregulation of major vault protein (MVP) expression after spinal cord injury in the brainstem of the adult zebrafish prompted us to probe for its contribution to recovery after SCI. MVP is a multifunctional protein expressed not only in many types of tumours but also in the nervous system, where its importance for regeneration is, however, unclear. Using an established zebrafish SCI model, we found that MVP mRNA and protein expression levels were increased in ependymal cells in the spinal cord caudal to the lesion site at 6 and 11 days after SCI. Double immunolabelling showed that MVP was co‐localised with Islet‐1 or tyrosine hydroxylase around the central canal of the spinal cord in sham‐injured control fish and injured fish 11 days after surgery. MVP co‐localised with the neural stem cell marker nestin in ependymal cells after injury. By using an in vivo morpholino‐based knock‐down approach, we found that the distance moved by MVP morpholino‐treated fish was reduced at 4, 5 and 6 weeks after SCI when compared to fish treated with standard control morpholino. Knock‐down of MVP resulted in reduced regrowth of axons from brainstem neurons into the spinal cord caudal to the lesion site. These results indicate that MVP supports locomotor recovery and axonal regrowth after SCI in adult zebrafish.     

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.     

Synaptic development in the injured spinal cord cavity following co-transplantation of fetal spinal cord cells and autologous activated Schwann cells

Transplantation of activated transgenic Schwann cells or a fetal spinal cord cell suspension has been widely used to treat spinal cord injury. However, little is known regarding the effects of co-transplantation. In the present study, autologous Schwann cells in combination with a fetal spinal cord cell suspension were transplanted into adult Wistar rats with spinal cord injury, and newly generated axonal connections were observed ultrastructurally. Transmission electron microscopic observations showed that...     

Anatomical studies of dorsal column axons and dorsal root ganglion cells after spinal cord injury in the newborn rat

The response of dorsal column axons was studied after neonatal spinal overhemisection injury (right hemicord and left doral funiculus). Rat pups (N = 11) received this spinal lesion at the C2 level within 30 hours after birth. The cauda equina was exposed 3 months later in one group of chronic operates (N = 5) and in a group of normal adults (N = 2), and all spinal roots from L5 caudally were cut bilaterally; 4 days later the spinal cord and medulla were processed for Fink-Heimer impregnation of degenerating axons and terminals. In a second group of chronic operates (N = 6) and normal adult controls (N = 4) the left sciatic nerve was injected with a cholera toxin-HRP conjugate (C-HRP), followed by a 2-3 day transganglionic transport period, and then the spinal cord and medulla were processed with tetramethylbenzidine histochemistry. Both control groups have a consistent dense projection in topographically adjacent regions of the dorsal funiculus and gracile nucleus. However, there is no sign of axonal growth around the lesion in either group of chronic experimental operates. Instead, there is a decreased density of projection within the dorsal funiculus near the lesion site. Many remaining C-HRP labeled axons in the experimental operates have abnormal, thick varicosities and swollen axonal endings (5-10 microns x 10-30 microns) within the dorsal funiculus through several spinal segments caudal to the lesion. Ultrastructural analysis of the dorsal funiculus in three other chronic experimental operates reveals the presence of numerous vesicle filled axonal profiles and reactive endings which appear similar to the C-HRP labeled structures. Transganglionic labeling after C-HRP sciatic nerve injections (N = 4) and retrograde labeling of L4, L5 dorsal root ganglion neurons after fast blue injections of the gracile nucleus (N = 6) both suggest that all dorsal column axons project to the gracile nucleus in the newborn rat. Dorsal root ganglion (DRG) cell survival following the neonatal overhemisection injury was also examined in the L4 and L5 DRG. DRG neurons that project to the gracile nucleus were prelabeled by injecting fast blue into this nucleus at birth two days prior to the cervical overhemisection spinal injury. Both normal littermates (N = 9) and spinally injured animals (N = 12) were examined after postinjection survival periods of 10 or 22 days.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

Suppression of astroglial scar formation and enhanced axonal regeneration associated with functional recovery in a spinal cord injury rat model by the cell cycle inhibitor olomoucine

It is well established that axons of the adult mammalian CNS are capable of regrowing only a limited amount after injury. Astrocytes are believed to play a crucial role in the failure to regenerate, producing multiple inhibitory proteoglycans, such as chondroitin sulphate proteoglycans (CSPGs). After spinal cord injury (SCI), astrocytes become hypertrophic and proliferative and form a dense network of astroglial processes at the site of lesion constituting a physical and biochemical barrier. Down-regulations of astroglial proliferation and inhibitory CSPG production might facilitate axonal regeneration. Recent reports indicated that aberrant activation of cell cycle machinery contributed to overproliferation and apoptosis of cells in various insults. In the present study, we sought to determine whether a cell cycle inhibitior, olomoucine, would decrease neuronal cell death, limit astroglial proliferation and production of inhibitory CSPGs, and eventually enhance the functional compensation after SCI in rats. Our results showed that up-regulations of cell cycle components were closely associated with neuronal cell death and astroglial proliferation as well as the production of CSPGs after SCI. Meanwhile, administration of olomoucine, a selective cell cycle kinase (CDK) inhibitor, has remarkably reduced the up-regulated cell cycle proteins and then decreased neuronal cell death, astroglial proliferation, and accumulation of CSPGs. More importantly, the treatment with olomoucine has also increased expression of growth-associated proteins-43, reduced cavity formation, and improved functional deficits. We consider that suppressing astroglial cell cycle in acute SCIs is beneficial to axonal growth. In the future, therapeutic strategies can be designed to achieve efficient axonal regeneration and functional compensation after traumatic CNS injury.     

Gamma-aminobutyric acid-containing sympathetic preganglionic neurons in rat thoracic spinal cord send their axons to the superior cervical ganglion

Gamma-aminobutyric acid (GABA)-containing fibers have been observed in the rat superior cervical ganglion (SCG) and, to a lesser extent, in the stellate ganglion (STG). The aim of present study is to clarify the source of these fibers. No cell body showed mRNAs for glutamic acid decarboxylases (GADs) or immunoreactivity for GAD of 67 kDa (GAD67) in the cervical sympathetic chain. Thus, GABA-containing fibers in the ganglia are suggested to be of extraganglionic origin. GAD67-immunoreactive fibers were found not in the dorsal roots or ganglia, but in the ventral roots, so GABA-containing fibers in the sympathetic ganglia were considered to originate from the spinal cord. Furthermore, almost all GAD67-immunoreactive fibers in the sympathetic ganglia showed immunoreactivity for vesicular acetylcholine transporter, suggesting that GABA was utilized by some cholinergic preganglionic neurons. This was confirmed by the following results. 1) After injection of Sindbis/palGFP virus into the intermediolateral nucleus, some anterogradely labeled fibers in the SCG were immunopositive for GAD67. 2) After injection of fluorogold into the SCG, some retrogradely labeled neurons in the thoracic spinal cord were positive for GAD67 mRNA. 3) When the ventral roots of the eighth cervical to the fourth thoracic segments were cut, almost all GAD67- and GABA-immunoreactive fibers disappeared from the ipsilateral SCG and STG, suggesting that the vast majority of GABA-containing fibers in those ganglia were of spinal origin. Thus, the present findings strongly indicate that some sympathetic preganglionic neurons are not only cholinergic but also GABAegic.