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Retrograde cell death in sensory dorsal root ganglion cells following peripheral nerve injury is well established. However, available data regarding the underlying mechanism behind injury induced motoneuron death are conflicting. By comparing morphological and molecular changes in spinal motoneurons after L4-L5 ventral root avulsion (VRA) and distal peripheral nerve axotomy (PNA) 7 and 14 days postoperatively, we aimed to gain more insight about the mechanism behind injury-induced motoneuron degeneration. Morphological changes in spinal cord were assessed by using quantitative immunohistochemistry. Neuronal degeneration was revealed by decreased immunostaining for microtubule-associated protein-2 in dendrites and synaptophysin in presynaptic boutons after both VRA and PNA. Significant motoneuron atrophy was already observed at 7 days post-injury, independently of injury type. Immunostaining for ED1 reactive microglia was significantly elevated in all experimental groups, as well as the astroglial marker glial fibrillary acidic protein (GFAP). Quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis of the ventral horn from L4-L5 spinal cord segments revealed a significant upregulation of genes involved in programmed cell death including caspase-3, caspase-8, and related death receptors TRAIL-R, tumor necrosis factor (TNF)-R, and Fas following VRA. In contrast, following PNA, caspase-3 and the death receptor gene expression levels did not differ from the control, and there was only a modest increased expression of caspase-8. Moreover, the altered gene expression correlated with protein changes. These results show that the spinal motoneurons reacted in a similar fashion with respect to morphological changes after both proximal and distal injury. However, the increased expression of caspase-3, caspase-8, and related death receptors after VRA suggest that injury- induced motoneuron degeneration is mediated through an apoptotic mechanism, which might involve both the intrinsic and the extrinsic pathways.

Peripheral nerve injuries induce significant sensory neuronal cell death in the dorsal root ganglia (DRG); however, the role of specific apoptotic pathways is still unclear. In this study, we performed peripheral nerve transection on adult rats, after which the corresponding DRGs were harvested at 7, 14, and 28 days after injury for subsequent molecular analyses with quantitative reverse transcription-PCR, western blotting, and immunohistochemistry. Nerve injury led to increased levels of caspase-3 mRNA and active caspase-3 protein in the DRG. Increased expression of caspase-8, caspase-12, caspase-7, and calpain suggested that both the extrinsic and the endoplasmic reticulum (ER) stress-mediated apoptotic pathways were activated. Phosphorylation of protein kinase R-like ER kinase further implied the involvement of ER-stress in the DRG. Phosphorylated protein kinase R-like ER kinase was most commonly associated with isolectin B4 (IB4)-positive neurons in the DRG and this may provide an explanation for the increased susceptibility of these neurons to die following nerve injury, likely in part because of an activation of the ER-stress response.

Introduction: Previously we showed that a fibrin glue conduit with human mesenchymal stem cells (hMSCs) and cyclosporine A (CsA) enhanced early nerve regeneration. In this study long term effects of this conduit are investigated. Methods: In a rat model, the sciatic nerve was repaired with fibrin conduit containing fibrin matrix, fibrin conduit containing fibrin matrix with CsA treatment and fibrin conduit containing fibrin matrix with hMSCs and CsA treatment, and also with nerve graft as control. Results: At 12 weeks 34% of motoneurons of the control group regenerated axons through the fibrin conduit. CsA treatment alone or with hMSCs resulted in axon regeneration of 67% and 64% motoneurons respectively. The gastrocnemius muscle weight was reduced in the conduit with fibrin matrix. The treatment with CsA or CsA with hMSCs induced recovery of the muscle weight and size of fast type fibers towards the levels of the nerve graft group. Discussion: The transplantation of hMSCs for peripheral nerve injury should be optimized to demonstrate their beneficial effects. The CsA may have its own effect on nerve regeneration.

The successful outcome of peripheral neuronal regeneration is attributed both to the growth permissive milieu and the intrinsic ability of the neuron to initiate appropriate cellular responses such as changes in gene expression and cytoskeletal rearrangements. Even though numerous studies have shown the importance of interactions between the neuron and the extracellular matrix (ECM) in axonal outgrowth, the molecular mechanisms underlying the contact between ECM receptors and the cellular cytoskeleton remain largely unknown. Unconventional myosins constitute an important group of cytoskeletal-associated motor proteins. One member of this family is the recently described myosin-X. This protein interacts with several members of the axon growth-associated ECM receptor family of integrins and could therefore be important in neuronal outgrowth. In this study, using radioactive in situ hybridization, we found that expression of myosin-X mRNA is upregulated in adult rat sensory neurons and spinal motoneurons after peripheral nerve injury, but not after central injury. Thus, myosin-X was upregulated after injuries that can be followed by axonal regeneration. We also found that the protein is localized to neuronal growth cones and that silencing of myosin-X using RNA interference impairs the integrin-mediated growth of neurites on laminin, but has no effect on non-integrin mediated growth on N-cadherin.

Olfactory ensheathing cells (OEC) have been shown to stimulate regeneration, myelination and functional recovery in different spinal cord injury models. However, recent reports from several laboratories have challenged this treatment strategy. The discrepancy in results could be attributed to many factors including variations in culture protocols. The present study investigates whether the differences in culture preparation could influence neuroprotective and growth-promoting effects of OEC after transplantation into the injured spinal cord. Primary OEC cultures were purified using method of differential cell adhesion (a-OEC) or separated with immunomagnetic beads (b-OEC). After cervical C4 hemisection in adult rats, short-term (3 weeks) or long-term (7 weeks) cultured OEC were transplanted into the lateral funiculus at 1mm rostral and caudal to the transection site. At 3-8 weeks after transplantation, labeled OEC were mainly found in the injection sites and in the trauma zone. Short-term cultured a-OEC supported regrowth of rubrospinal, raphaespinal and CGRP-positive fibers, and attenuated retrograde degeneration in the red nucleus. Short-term cultured b-OEC failed to promote axonal regrowth but increased the density of rubrospinal axons within the dorsolateral funiculus and provided significant neuroprotection for axotomized rubrospinal neurons. In addition, short-term cultured OEC attenuated sprouting of rubrospinal terminals. In contrast, long-term cultured OEC neither enhanced axonal growth nor prevented retrograde cell death. The results suggest that the age of OEC in culture and the method of cell purification could affect the efficacy of OEC to support neuronal survival and regeneration after spinal cord injury. This article is part of a Special Issue entitled: Understanding olfactory ensheathing glia and their prospect for nervous system repair.

Peripheral nerve injuries are often associated with loss of nerve tissue and require autologous nerve grafts to provide a physical substrate for axonal growth. Biosynthetic neural conduits could be an alternative treatment strategy in such injuries. The present study investigates the long-term effects of a tubular fibrin conduit on neuronal regeneration, axonal sprouting and recovery of muscle weight following peripheral nerve injury and repair in adult rats. Sciatic axotomy was performed proximally in the thigh to create a 10-mm gap between the nerve stumps. The injury gap was bridged by using a 14-mm-long fibrin glue conduit, entubulating 2 mm of the nerve stump at each end. A reversed autologous nerve graft was used as a control. The regenerative response from sensory and motor neurones was evaluated following retrograde labelling with Fast Blue fluorescent tracer. In control experiments, at 16 weeks following peripheral nerve grafting, 5184 (±574 standard error of mean (SEM)) sensory dorsal root ganglion neurones and 1001 (±37 SEM) spinal motor neurones regenerated across the distal nerve-graft interface. The fibrin conduit promoted regeneration of 60% of sensory neurones and 52% of motor neurones when compared to the control group. The total number of myelinated axons in the distal nerve stump in the fibrin-conduit group reached 86% of the control and the weight of gastrocnemius and soleus muscles recovered to 82% and 89% of the controls, respectively. The present results suggest that a tubular fibrin conduit can be used to promote neuronal regeneration following peripheral nerve injury.

Despite surgical innovation, the sensory and motor outcome after a peripheral nerve injury remains incomplete. One contributing factor to the poor outcome is prolonged denervation of the target organ, leading to apoptosis of both mature myofibres and satellite cells with subsequent replacement of the muscle tissue with fibrotic scar and adipose tissue. In this study, we investigated the expression of myogenic transcription factors, muscle specific microRNAs and muscle-specific E3 ubiquitin ligases at several time points following denervation in two different muscles, the gastrocnemius (containing predominantly fast type fibres) and soleus (slow type) muscles, since these molecules may influence the degree of atrophy following denervation. Both muscles exhibited significant atrophy (compared with the contra-lateral sides) at 7 days following either a nerve transection or crush injury. In the crush model, the soleus muscle showed significantly increased muscle weights at days 14 and 28 which was not the case for the gastrocnemius muscle which continued to atrophy. There was a significantly more pronounced up-regulation of MyoD expression in the denervated soleus muscle compared with the gastrocnemius muscle. Conversely, myogenin was more markedly elevated in the gastrocnemius versus soleus muscles. The muscles also showed significantly contrasting transcriptional regulation of the microRNAs miR-1 and miR-206. MuRF1 and Atrogin-1 showed the highest levels of expression in the denervated gastrocnemius muscle. This study provides further insights regarding the intracellular regulatory molecules that generate and maintain distinct patterns of gene expression in different fibre types following peripheral nerve injury.

Integrins are cell surface receptors known to be important for regeneration in the peripheral nervous system. We have investigated the expression of integrin messenger RNAs in red nucleus neurons of adult rats after axotomy and administration of neurotrophic factors. Using radioactive in situ hybridization, messenger RNA for integrin subunits beta1, alpha3, alpha7 and alphaV could be detected. No change of any alpha subunit could be detected after axotomy. In contrast, a small upregulation of beta1 was detected after lesion. Administration of neurotrophin-3 induced a robust further increase in beta1 messenger RNA levels, whereas brain-derived neurotrophic factor did not. By analogy to the peripheral nervous system, we propose that integrins may be important for a regenerative response in central nervous system neurons.

Spinal cord injury (SCI) induces retrograde cell death in descending pathways, which can be prevented by long-term intrathecal infusion of neurotrophins (Novikova et al. [2000] Eur J Neurosci 12:776-780). The present study investigates whether the same treatment also leads to improved regeneration of the injured tracts. After cervical SCI in adult rats, a peripheral nerve graft was attached to the rostral wall of the lesion cavity. The animals were treated by local application into the cavity of Gelfoam soaked in (1) phosphate buffered saline (untreated controls) or (2) a mixture of the neurotrophins brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) (local treatment), or by intrathecal infusion of BDNF + NT-3 for (3) 2 weeks (short-term treatment) or (4) 5-8 weeks (long-term treatment). Despite a very strong survival effect, long-term treatment failed to stimulate ingrowth of descending tracts into the nerve graft. In comparison with untreated controls, the latter treatment also caused 35% reduction in axonal sprouting of descending pathways rostral to the lesion site and 72% reduction in the number of spinal cord neurons extending axons into the nerve graft. Local and short-term treatments neither prevented retrograde cell death nor enhanced regeneration of descending tracts, but induced robust regeneration of spinal cord neurons into the nerve graft. These results indicate that the signal pathways promoting neuronal survival and axonal regeneration, respectively, in descending tracts after SCI respond differently to neurotrophic stimuli and that efficient rescue of axotomized tract neurons is not a sufficient prerequisite for regeneration.

Regeneration of dorsal roots of spinal nerves in rats after transplantation of embryonic nerve tissue.

Spinal cord injury triggers a cascade of degenerative changes leading to cell death and cavitation. Severed axons fail to regenerate across the scar tissue and are only capable of limited sprouting. In this study, we investigated the effects of adult human adipose-derived stem cells (ASC) on axonal regeneration following transplantation into the injured rat cervical spinal cord. ASC did not induce activation of astrocytes in culture and supported neurite outgrowth from adult rat sensory dorsal root ganglia neurons. After transplantation into the lateral funiculus 1 mm rostral and caudal to the cervical C3-C4 hemisection, ASC continued to express brain-derived neurotrophic factor, vascular endothelial growth factor, and fibroblast growth factor-2 for 3 weeks but only in animals treated with cyclosporine A. Transplanted ASC stimulated extensive ingrowth of 5HT-positive raphaespinal axons into the trauma zone with some terminal arborizations reaching the caudal spinal cord. In addition, ASC induced sprouting of raphaespinal terminals in C2 contralateral ventral horn and C6 ventral horn on both sides. Transplanted cells also changed the structure of the lesion scar with numerous astrocytic processes extended into the middle of the trauma zone in a chain-like pattern and in close association with regenerating axons. The density of the astrocytic network was also significantly decreased. Although the transplanted cells had no effect on the density of capillaries around the lesion site, the activity of OX42-positive microglial cells was markedly reduced. However, ASC did not support recovery of forelimb function. The results suggest that transplanted ASC can modify the structure of the glial scar and stimulate axonal sprouting.

Brain-derived neurotrophic factor has previously been shown to promote survival and axonal regeneration in injured spinal motoneurons and, also, to modulate synaptic transmission and regulate the density of synaptic innervation in a variety of neurons. The present light and electron microscopic study demonstrates synaptotrophic effects of exogenously applied brain-derived neurotrophic factor on the synaptic composition of both normal and axonally lesioned adult rat spinal motoneurons. After L5-L6 ventral root avulsion, a massive loss of all types of boutons occurred on the somata of the lesioned motoneurons which persisted for at least 12 weeks postoperatively. We found that (i) intrathecal infusion of brain-derived neurotrophic factor during the first postoperative week did not prevent the synaptic detachment and activation of glial cells; (ii) prolonged treatment for four weeks restored synaptic covering and significantly reduced microglial reaction; (iii) the synaptotrophic effect remained significant for at least eight weeks after cessation of the treatment; (iv) brain-derived neurotrophic factor mainly supported F-type boutons with presumably inhibitory function, while it had little effect on S-type boutons associated with excitatory action; and (v) in normal unlesioned motoneurons, four weeks of treatment with brain-derived neurotrophic factor induced sprouting of F-type boutons, a loss of S-type boutons and motoneuron atrophy. The present data show that exogenous neurotrophins not only help to restore synaptic circuitry in axonally injured motoneurons, but also strongly influence the synaptic composition in normal motoneurons.

This study shows that both BDNF and NT-3 can prevent cell death in axotomized adult rat rubrospinal neurons (RSNs), but that the efficacy of neuroprotection depends on the temporal pattern of treat ...

Neurotrophin-3 (NT-3) and brain-derived neurotrophic factor (BDNF) have previously been shown to support survival and axonal regeneration in various types of neurons. Also, synergistic neuroprotective effects of these neurotrophins have been reported in descending rubrospinal neurons after cervical spinal cord injury (Novikova et al., [2000] Eur. J. Neurosci. 12:776-780). The present study investigates the effects of intrathecally delivered NT-3 and BDNF on the survival and atrophy of ascending spinocerebellar neurons of Clarke nucleus (CN) after cervical spinal cord injury in adult rats. At 8 weeks after cervical spinal cord hemisection, 40% of the axotomized CN neurons had been lost, and the remaining cells exhibited marked atrophy. Microglial activity was significantly increased in CN of the operated side. Intrathecal infusion of NT-3 for 8 weeks postoperatively resulted in 91% cell survival and a reduction in cell atrophy, but did not reduce microglial activity. In spite of the fact that the CN neurons expressed both TrkC and TrkB receptors, only NT-3 had a neuroprotective effect, whereas BDNF was ineffective. Furthermore, when a combination of BDNF and NT-3 was administered, the neuroprotective effect of NT-3 was lost. The present results indicate a therapeutic potential for NT-3 in the treatment of spinal cord injury, but also demonstrate that in certain neuronal populations the neuroprotection obtained by a combination of neurotrophic factors may be less than that of a single neurotrophin.

This study shows that in adult rat spinal motoneurons brain-derived neurotrophic factor exerts a neuroprotective effect which extends several weeks beyond the duration of treatment. In addition, brain-derived neurotrophic factor strongly enhances regeneration of avulsed motor axons across the border between the central and peripheral nervous systems. Treatment with brain-derived neurotrophic factor is known to rescue adult rat spinal motoneurons from retrograde cell death induced by ventral root avulsion. The present experiments were designed to test whether this survival effect remains over an extended period of time following cessation of treatment and, also, whether brain-derived neurotrophic factor promotes regeneration of avulsed motor axons. After avulsion of a spinal ventral root, four weeks of treatment with brain-derived neurotrophic factor (10 microg/day) or vehicle was initiated. By using different retrograde tracers to obtain pre- and postoperative labelling of avulsed and regenerating motoneurons, respectively, the number of surviving motoneurons as well as the extent of motor axonal regeneration could be analysed. The expression of nitric oxide synthase in the lesioned motoneurons was also studied. In the vehicle-treated rats, only 10% of the avulsed motoneurons remained at 12 weeks postoperatively, 20-40% of which displayed nitric oxide synthase activity. Treatment with brain-derived neurotrophic factor during the initial four postoperative weeks resulted in 45% motoneuron survival and a complete blockage of nitric oxide synthase expression at 12 weeks postoperatively. Brain-derived neurotrophic factor also induced abundant regeneration of the avulsed motor axons, which formed extensive fibre bundles along the surface of the spinal cord and adjacent ventral roots. The long-term effect by brain-derived neurotrophic factor seemed to be even stronger on motor axonal regeneration than on motoneuron survival. The present results indicate a therapeutic potential for brain-derived neurotrophic factor in the early treatment of traumatic injuries to spinal nerves and roots.

In future, adipose-derived stem cells (ASC) might be used to treat neurological disorders. In this study, the neurotrophic and angiogenic properties of human ASC were evaluated, and their effects in a peripheral nerve injury model were determined. In vitro growth factor stimulation of the cells resulted in increased secretion of brain-derived neurotrophic factor (BDNF), glial cell-derived neurotrophic factor (GDNF), vascular endothelial growth factor-A (VEGF-A), and angiopoietin-1 proteins. Conditioned medium from stimulated cells increased neurite outgrowth of dorsal root ganglia (DRG) neurons. Similarly, stimulated cells showed an enhanced ability to induce capillary-like tube formation in an in vitro angiogenesis assay. ASC were seeded into a fibrin conduit that was used to bridge a 10 mm rat nerve gap. After 2 weeks, the animals treated with control or stimulated ASC showed an enhanced axon regeneration distance. Stimulated cells evoked more total axon growth. Analysis of regeneration and apoptosis-related gene expression showed that both ASC and stimulated ASC enhanced GAP-43 and activating transcription factor 3 (ATF-3) expression in the spinal cord and reduced c-jun expression in the DRG. Caspase-3 expression in the DRG was reduced by stimulated ASC. Both ASC and stimulated ASC also increased the vascularity of the fibrin nerve conduits. Thus, ASC produce functional neurotrophic and angiogenic factors, creating a more desirable microenvironment for nerve regeneration.

Despite advances in surgical techniques for peripheral nerve repair, functional restitution remains incomplete. The timing of surgery is one factor influencing the extent of recovery but it is not yet clearly defined how long a delay may be tolerated before repair becomes futile. In this study, rats underwent sciatic nerve transection before immediate (0) or 1, 3, or 6 months delayed repair with a nerve graft. Regeneration of spinal motoneurons, 13 weeks after nerve repair, was assessed using retrograde labeling. Nerve tissue was also collected from the proximal and distal stumps and from the nerve graft, together with the medial gastrocnemius (MG) muscles. A dramatic decline in the number of regenerating motoneurons and myelinated axons in the distal nerve stump was observed in the 3- and 6-months delayed groups. After 3 months delay, the axonal number in the proximal stump increased 2-3 folds, accompanied by a smaller axonal area. RT-PCR of distal nerve segments revealed a decline in Schwann cells (SC) markers, most notably in the 3 and 6 month delayed repair samples. There was also a progressive increase in fibrosis and proteoglycan scar markers in the distal nerve with increased delayed repair time. The yield of SC isolated from the distal nerve segments progressively fell with increased delay in repair time but cultured SC from all groups proliferated at similar rates. MG muscle at 3- and 6-months delay repair showed a significant decline in weight (61% and 27% compared with contra-lateral side). Muscle fiber atrophy and changes to neuromuscular junctions were observed with increased delayed repair time suggestive of progressively impaired reinnervation. This study demonstrates that one of the main limiting factors for nerve regeneration after delayed repair is the distal stump. The critical time point after which the outcome of regeneration becomes too poor appears to be 3-months.

Clinical efficacy of stem cells for nerve repair is likely to be influenced by issues including donor age and in vitro expansion time. We isolated human mesenchymal stem cells (MSC) from bone marrow of young (16–18 years) and old (67–75 years) donors and analyzed their capacity to differentiate and promote neurite outgrowth from dorsal root ganglia (DRG) neurons. Treatment of MSC with growth factors (forskolin, basic fibroblast growth factor, platelet derived growth factor-AA and glial growth factor-2) induced protein expression of the glial cell marker S100 in cultures from young but not old donors. MSC expressed various neurotrophic factor mRNA transcripts. Growth factor treatment enhanced the levels of BDNF and VEGF transcripts with corresponding increases in protein release in both donor cell groups. MSC in co-culture with DRG neurons significantly enhanced total neurite length which, in the case of young but not old donors, was further potentiated by treatment of the MSC with the growth factors. Stem cells from young donors maintained their proliferation rate over a time course of 9 weeks whereas those from the old donors showed increased population doubling times. MSC from young donors, differentiated with growth factors after long-term culture, maintained their ability to enhance neurite outgrowth of DRG. Therefore, MSC isolated from young donors are likely to be a favourable cell source for nerve repair.

To address the need for the development of bioengineered replacement of a nerve graft, a novel two component fibrin glue conduit was combined with human mesenchymal stem cells (MSC) and immunosupressive treatment with cyclosporine A. The effects of MSC on axonal regeneration in the conduit and reaction of activated macrophages were investigated using sciatic nerve injury model. A 10mm gap in the sciatic nerve of a rat was created and repaired either with fibrin glue conduit containing diluted fibrin matrix or fibrin glue conduit containing fibrin matrix with MSC at concentration of 80×10(6) cells/ml. Cells were labeled with PKH26 prior to transplantation. The animals received daily injections of cyclosporine A. After 3 weeks the distance of regeneration and area occupied by regenerating axons and ED1 positives macrophages was measured. MSC survived in the conduit and enhanced axonal regeneration only when transplantation was combined with cyclosporine A treatment. Moreover, addition of cyclosporine A to the conduits with transplanted MSC significantly reduced the ED1 macrophage reaction.

Peripheral nerve injuries with loss of nervous tissue are a significant clinical problem and are currently treated using autologous nerve transplants. To avoid the need for donor nerve, which results in additional morbidity such as loss of sensation and scarring, alternative bridging methods have been sought. Recently we showed that an artificial nerve conduit moulded from fibrin glue is biocompatible to nerve regeneration. In this present study, we have used the fibrin conduit or a nerve graft to bridge either a 10 mm or 20 mm sciatic nerve gap and analyzed the muscle recovery in adult rats after 16 weeks. The gastrocnemius muscle weights of the operated side were similar for both gap sizes when treated with nerve graft. In contrast, muscle weight was 48.32 ± 4.96% of the contra-lateral side for the 10 mm gap repaired with fibrin conduit but only 25.20 ± 2.50% for the 20 mm gap repaired with fibrin conduit. The morphology of the muscles in the nerve graft groups showed an intact, ordered structure, with the muscle fibers grouped in fascicles whereas the 20 mm nerve gap fibrin group had a more chaotic appearance. The mean area and diameter of fast type fibers in the 20 mm gap repaired with fibrin conduits were significantly (P0.01) worse than those of the corresponding 10 mm gap group. In contrast, both gap sizes treated with nerve graft showed similar fiber size. Furthermore, the 10 mm gaps repaired with either nerve graft or fibrin conduit showed similar muscle fiber size. These results indicate that the fibrin conduit can effectively treat short nerve gaps but further modification such as the inclusion of regenerative cells may be required to attain the outcomes of nerve graft for long gaps.

Development of cell-based treatment strategies for repair of the injured nervous system requires cell tracing techniques to follow the fate of transplanted cells and their interaction with the host tissue. The present study investigates the efficacy of fluorescent cell tracers Fast Blue, PKH26, DiO and CMFDA for long-term labeling of olfactory ensheathing glial cells (OEC) in culture and following transplantation into the rat spinal cord. All tested dyes produced very efficient initial labeling of p75-positive OEC in culture. The number of Fast Blue-positive cells remained largely unchanged during the first 4 weeks but only about 21% of the cells retained tracer 6 weeks after labeling. In contrast, the number of cells labeled with PKH26 and DiO was reduced to 51-55% after 2 weeks in culture and reached 8-12% after 4-6 weeks. CMFDA had completely disappeared from the cells 2 weeks after labeling. AlamarBlue assay showed that among four tested tracers only CMFDA reduced proliferation rate of the OEC. After transplantation into spinal cord, Fast Blue-labeled OEC survived for at least 8 weeks but demonstrated very limited migration from the injection sites. Additional immunostaining with glial and neuronal markers revealed signs of dye leakage from the transplanted cells resulted in weak labeling of microglia and spinal neurons. The results show that Fast Blue is an efficient cell marker for cultured OEC. However, transfer of the dye from the transplanted cells to the host tissue should be considered and correctly interpreted.

Microsurgical reconstruction of injured peripheral nerves often results in limited functional recovery. One contributing factor is the retrograde neuronal degeneration of sensory neurons in the dorsal root ganglia (DRG) and of motor neurons in the spinal cord. The present study investigates the neuroprotective and growth-promoting effects of N-acetyl-cysteine (NAC) on sensory DRG neurons and spinal motoneurons after sciatic axotomy and nerve grafting in adult rats. Sciatic axotomy and nerve grafting were performed at 1 week after sural DRG neurons and motoneurons were retrogradely labeled with the fluorescent tracer Fast Blue. To assess the efficacy of axonal regeneration, a second fluorescent dye Fluoro-Ruby was applied distal to the graft at 12 weeks after nerve repair. At 8-13 weeks after axotomy, only 52-56% of the sural sensory neurons remained in the lumbar DRG, while the majority of motoneurons survived the sciatic nerve injury. Nerve grafting alone or continuous intrathecal NAC treatment (2.4 mg/day) improved survival of sural DRG neurons. Combined treatment with nerve graft and NAC had significant additive effect on neuronal survival and also increased the number of sensory neurons regenerating across the graft. However, NAC treatment neither affected the number of regenerating motoneurons nor the number of myelinated axons in the nerve graft or in the distal nerve stump. The present results demonstrate that NAC provides a highly significant effect of neuroprotection in an animal nerve injury model and that combination with nerve grafting further attenuates retrograde cell death and promotes regeneration of sensory neurons.

Development of biosynthetic conduits carrying extracellular matrix molecules and cell lines expressing neurotrophic growth factors represents a novel and promising strategy for spinal cord and peripheral nerve repair. In the present in vitro study, the compatibility and growth-promoting effects of (i) alginate hydrogel, (ii) alginate hydrogel complemented with fibronectin, and (iii) matrigel were compared between olfactory ensheathing cells (OECs), Schwann cells (SCs), and bone marrow stromal cells (BMSCs). Neurite outgrowth from embryonic dorsal root ganglia (DRG) neurons was used to assess the efficacy of the hydrogels alone or in combination with cultured cells to promote axonal regeneration. The result showed that alginate hydrogel transformed OECs, SCs, and BMSCs into atypical cells with spherical shape and inhibited their metabolic activity. Combination of alginate hydrogel with fibronectin promoted only OECs proliferation. Alginate hydrogel also inhibited outgrowth of DRG neurites, although this effect was attenuated by addition of fibronectin, SCs, or BMSCs. In contrast, matrigel stimulated cell proliferation, preserved the typical morphological features of the cultured cells and induced massive sprouting of DRG neurites. Addition of cultured cells to matrigel did not further improve DRG neurite outgrowth. The present findings suggest that addition of extracellular matrix should be considered when engineering biosynthetic scaffolds on the basis of alginate hydrogels.

After spinal cord injury, the severed neuronal pathways fail to regenerate spontaneously. This study describes a biodegradable implant using poly-beta-hydroxybutyrate (PHB) fibers as carrier scaffold for matrix components and cell lines supporting neuronal survival and regeneration after spinal cord injury. After cervical spinal cord injury in adult rats, a graft consisting of PHB fibers coated with alginate hydrogel + fibronectin was implanted in the lesion cavity. In control groups, PHB was omitted and only alginate hydrogel or fibronectin, or their combination, were used for grafting. In addition, comparisons were made with animals treated intrathecally after spinal cord injury with the neurotrophic factors BDNF or NT-3. The neurons of the rubrospinal tract served as experimental model. In untreated animals, 45% of the injured rubrospinal neurons were lost at 8 weeks postoperatively. Implantation of the PHB graft reduced this cell loss by 50%, a rescuing effect similar to that obtained after treatment with BDNF or NT-3. In the absence of PHB support, implants of only alginate hydrogel or fibronectin, or their combination, had no effect on neuronal survival. After addition of neonatal Schwann cells to the PHB graft, regenerating axons were seen to enter the graft from both ends and to extend along its entire length. These results show that implants using PHB as carrier scaffold and containing alginate hydrogel, fibronectin and Schwann cells can support neuronal survival and regeneration after spinal cord injury

Following the initial acute stage of spinal cord injury, a cascade of cellular and inflammatory responses will lead to progressive secondary damage of the nerve tissue surrounding the primary injury site. The degeneration is manifested by loss of neurons and glial cells, demyelination and cyst formation. Injury to the mammalian spinal cord results in nearly complete failure of the severed axons to regenerate. We have previously demonstrated that the antioxidants N-acetyl-cysteine (NAC) and acetyl-L-carnitine (ALC) can attenuate retrograde neuronal degeneration after peripheral nerve and ventral root injury. The present study evaluates the effects of NAC and ALC on neuronal survival, axonal sprouting and glial cell reactions after spinal cord injury in adult rats. Tibial motoneurons in the spinal cord were pre-labeled with fluorescent tracer Fast Blue one week before lumbar L5 hemisection. Continuous intrathecal infusion of NAC (2.4 mg/day) or ALC (0.9 mg/day) was initiated immediately after spinal injury using Alzet 2002 osmotic minipumps. Neuroprotective effects of treatment were assessed by counting surviving motoneurons and by using quantitative immunohistochemistry and Western blotting for neuronal and glial cell markers 4 weeks after hemisection. Spinal cord injury induced significant loss of tibial motoneurons in L4-L6 segments. Neuronal degeneration was associated with decreased immunostaining for microtubular-associated protein-2 (MAP2) in dendritic branches, synaptophysin in presynaptic boutons and neurofilaments in nerve fibers. Immunostaining for the astroglial marker GFAP and microglial marker OX42 was increased. Treatment with NAC and ALC rescued approximately half of the motoneurons destined to die. In addition, antioxidants restored MAP2 and synaptophysin immunoreactivity. However, the perineuronal synaptophysin labeling was not recovered. Although both treatments promoted axonal sprouting, there was no effect on reactive astrocytes. In contrast, the microglial reaction was significantly attenuated. The results indicate a therapeutic potential for NAC and ALC in the early treatment of traumatic spinal cord injury.