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Spinal cord injury results in irreversible tissue damage and permanent sensorimotor impairment. The development of novel therapeutic strategies that improve the life quality of affected individuals is therefore of paramount importance. Cell transplantation is a promising approach for spinal cord injury treatment and the present study assesses the efficacy of human embryonic stem cell–derived neural crest cells as preclinical cell-based therapy candidates. The differentiated neural crest cells exhibited characteristic molecular signatures and produced a range of biologically active trophic factors that stimulated in vitro neurite outgrowth of rat primary dorsal root ganglia neurons. Transplantation of the neural crest cells into both acute and chronic rat cervical spinal cord injury models promoted remodeling of descending raphespinal projections and contributed to the partial recovery of forelimb motor function. The results achieved in this proof-of-concept study demonstrates that human embryonic stem cell–derived neural crest cells warrant further investigation as cell-based therapy candidates for the treatment of spinal cord injury.

Traumatic injury to the central nervous system (CNS) is further complicated by an increase in secondary neuronal damage imposed by activated microglia/macrophages. MicroRNA-124 (miR-124) is responsible for mouse monocyte quiescence and reduction of their inflammatory cytokine production. We describe the formulation and ex vivo transfection of chitosan/miR-124 polyplex particles into rat microglia and the resulting reduction of reactive oxygen species (ROS) and TNF-α and lower expression of MHC-II. Upon microinjection into uninjured rat spinal cords, particles formed with Cy3-labeled control sequence RNA, were specifically internalized by OX42 positive macrophages and microglia cells. Alternatively particles injected in the peritoneum were transported by macrophages to the site of spinal cord injury 72h post injection. Microinjections of chitosan/miR-124 particles significantly reduced the number of ED-1 positive macrophages in the injured spinal cord. Taken together, these data present a potential treatment technique to reduce inflammation for a multitude of CNS neurodegenerative conditions.From the Clinical EditorThe treatment of spinal cord injury remains an unresolved problem. Secondary damage is often the result of inflammation caused by activated microglia and/or macrophages. In this article, the authors developed their formulation of chitosan/miR-124 polyplex particles and investigated their use in the suppression of neuronal inflammation. This exciting data may provide a new horizon for patients who suffer from spinal cord injury.

Peripheral nerve injuries result in severe loss of sensory and motor functions in the afflicted limb. There is a lack of standardised models to non-invasively study degeneration, regeneration, and normalisation of neuronal microstructure in peripheral nerves. This study aimed to develop a non-invasive evaluation of peripheral nerve injuries, using diffusion tensor imaging (DTI), diffusion kurtosis imaging (DKI), and tractography on a rat model of sciatic nerve injury. 10 female Sprague Dawley rats were exposed to sciatic nerve neurotmesis and studied using a 9.4 T magnet, by performing DTI and DKI of the sciatic nerve before and 4 weeks after injury. The distal nerve stump showed a decrease in fractional anisotropy (FA), mean kurtosis (MK), axonal water fraction (AWF), and radial and axonal kurtosis (RK, AK) after injury. The proximal stump showed a significant decrease in axial diffusivity (AD) and increase of MK and AK as compared with the uninjured nerve. Both mean diffusivity (MD) and radial diffusivity (RD) increased in the distal stump after injury. Tractography visualised the sciatic nerve and the site of injury, as well as local variations of the diffusion parameters following injury. In summary, the described method detects changes both proximal and distal to the nerve injury.

Surgical intervention is the current gold standard treatment following peripheral nerve injury. However, this approach has limitations, and full recovery of both motor and sensory modalities often remains incomplete. The development of artificial nerve grafts that either complement or replace current surgical procedures is therefore of paramount importance. An essential component of artificial grafts is biodegradable conduits and transplanted cells that provide trophic support during the regenerative process. Neural crest cells are promising support cell candidates because they are the parent population to many peripheral nervous system lineages. In this study, neural crest cells were differentiated from human embryonic stem cells. The differentiated cells exhibited typical stellate morphology and protein expression signatures that were comparable with native neural crest. Conditioned media harvested from the differentiated cells contained a range of biologically active trophic factors and was able to stimulate in vitro neurite outgrowth. Differentiated neural crest cells were seeded into a biodegradable nerve conduit, and their regeneration potential was assessed in a rat sciatic nerve injury model. A robust regeneration front was observed across the entire width of the conduit seeded with the differentiated neural crest cells. Moreover, the up‐regulation of several regeneration‐related genes was observed within the dorsal root ganglion and spinal cord segments harvested from transplanted animals. Our results demonstrate that the differentiated neural crest cells are biologically active and provide trophic support to stimulate peripheral nerve regeneration. Differentiated neural crest cells are therefore promising supporting cell candidates to aid in peripheral nerve repair.

Spinal cord injury (SCI) is often associated with scarring and cavity formation and therefore bridging strategies are essential to provide a physical substrate for axonal regeneration. In this study we investigated the effects of a biodegradable conduit made from trimethylene carbonate and ε-caprolactone (TC) containing poly-p-dioxanone microfilaments (PDO) with longitudinal grooves on regeneration after SCI in adult rats. In vitro studies demonstrated that different cell types including astrocytes, meningeal fibroblasts, Schwann cells and adult sensory dorsal root ganglia neurons can grow on the TC and PDO material. For in vivo experiments, the TC/PDO conduit was implanted into a small 2-3 mm long cavity in the C3-C4 cervical segments immediately after injury (acute SCI) or at 2-5 months after initial surgery (chronic SCI). At 8 weeks after implantation into acute SCI, numerous 5HT-positive descending raphaespinal axons and sensory CGRP-positive axons regenerated across the conduit and were often associated with PDO microfilaments and migrated host cells. Implantation into chronically injured SCI induced regeneration mainly of the sensory CGRP-positive axons. Although the conduit had no effect on the density of OX42-positive microglial cells when compared with SCI control, the activity of GFAP-positive astrocytes was reduced. The results suggest that a TC/PDO conduit can support axonal regeneration after acute and chronic SCI even without addition of exogenous glial or stem cells.Biosynthetic conduits can support regeneration after spinal cord injury but often require addition of cell therapy and neurotrophic factors. This study demonstrates that biodegradable conduits made from trimethylene carbonate and ε-caprolactone with poly-p-dioxanone microfilaments alone can promote migration of different host cells and stimulate axonal regeneration after implantation into acute and chronic spinal cord injury. These results can be used to develop biosynthetic conduits for future clinical applications.

Adipose derived stem cells (ADSC) can be differentiated into Schwann cell-like cells which enhance nerve function and regeneration. However, the signalling mechanisms underlying the neurotrophic potential of ADSC remain largely unknown. In this study, we hypothesised that ADSC, upon stimulation with a combination of growth factors, could rapidly produce brain derived neurotrophic factor (BDNF) with a similar molecular mechanism to that functioning in the nervous system. Within 48 h of stimulation, ADSC demonstrated potent neurotrophic effects on dorsal root ganglion neurons, at a magnitude equivalent to that of the longer term differentiated Schwann cell-like cells. Stimulated ADSC showed rapid up-regulation of the neuronal activity dependent promoter BDNF exon IV along with an augmented expression of full length protein encoding BDNF exon IX. BDNF protein was secreted at a concentration similar to that produced by differentiated Schwann cell-like cells. Stimulation also activated the BDNF expression gating transcription factor, cAMP responsive element binding (CREB) protein. However, blocking phosphorylation of CREB with the protein kinase A small molecule inhibitor H89 did not suppress secretion of BDNF protein. These results suggest rapid BDNF production in ADSC is mediated through multiple compensatory pathways independent of, or in addition to, the CREB neuronal activation cascade.

Traumatic spinal cord injury induces a long-standing inflammatory response in the spinal cord tissue, leading to a progressive apoptotic death of spinal cord neurons and glial cells. We have recently demonstrated that immediate treatment with the antioxidants N-acetyl-cysteine (NAC) and acetyl-l-carnitine (ALC) attenuates neuroinflammation, induces axonal sprouting, and reduces the death of motoneurons in the vicinity of the trauma zone 4weeks after initial trauma. The objective of the current study was to investigate the effects of long-term antioxidant treatment on the survival of descending rubrospinal neurons after spinal cord injury in rats. It also examines the short- and long-term effects of treatment on apoptosis, inflammation, and regeneration in the spinal cord trauma zone. Spinal cord hemisection performed at the level C3 induced a significant loss of rubrospinal neurons 8 weeks after injury. At 2 weeks, an increase in the expression of the apoptosis-associated markers BCL-2-associated X protein (BAX) and caspase 3, as well as the microglial cell markers OX42 and ectodermal dysplasia 1 (ED1), was seen in the trauma zone. After 8 weeks, an increase in immunostaining for OX42 and the serotonin marker 5HT was detected in the same area. Antioxidant therapy reduced the loss of rubrospinal neurons by approximately 50%. Treatment also decreased the expression of BAX, caspase 3, OX42 and ED1 after 2 weeks. After 8 weeks, treatment decreased immunoreactivity for OX42, whereas it was increased for 5HT. In conclusion, this study provides further insight in the effects of treatment with NAC and ALC on descending pathways, as well as short- and long-term effects on the spinal cord trauma zone.

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.

Brachial plexus injury (BPI) is a devastating type of nerve injury, potentially causing loss of motor and sensory function. Principally, BPI is either categorized as preganglionic or post- ganglionic, with the early establishment of injury level being crucial for choosing the correct treatment strategy. Despite diagnostic advances, the need for a reliable, non-invasive method for establishing the injury level remains. We studied the usefulness of in vivo mag- netic resonance imaging (MRI) of the spinal cord for determination of injury level. The find- ings were related to neuronal and glial changes. Rats underwent unilateral L4 & L5 ventral roots avulsion or sciatic nerve axotomy. The injuries served as models for pre- and postgan- glionic BPI, respectively. MRI of the L4/L5 spinal cord segments 4 weeks after avulsion showed ventral horn (VH) shrinkage on the injured side compared to the uninjured side. Axotomy induced no change in the VH size on MRI. Following avulsion, histological sections of L4/L5 revealed shrinkage in the VH grey matter area occupied by NeuN-positive neurons, loss of microtubular-associated protein-2 positive dendritic branches (MAP2), pan-neurofila- ment positive axons (PanNF), synaptophysin-positive synapses (SYN) and increase in immunoreactivity for the microglial OX42 and astroglial GFAP markers. Axotomy induced no changes in NeuN-reactivity, modest decrease of MAP2 immunoreactivity, no changes in SYN and PanNF labelling, and a modest increase in OX42 and SYN labeling. Histological and radiological findings were congruent when assessing changes after axotomy, while MRI somewhat underestimated the shrinkage. This study indicates a potential diagnostic value of structural spinal cord MRI following BPI.

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.

This study investigated the effects of a membrane conduit filled with a synthetic matrix BD™ PuraMatrix™ peptide (BD) hydrogel and cultured Schwann cells on regeneration after peripheral nerve injury in adult rats. After sciatic axotomy, a 10mm gap between the nerve stumps was bridged using ultrafiltration membrane conduits filled with BD hydrogel or BD hydrogel containing Schwann cells. In control experiments, the nerve defect was bridged using either membrane conduits with alginate/fibronectin hydrogel or autologous nerve graft. Axonal regeneration within the conduit was assessed at 3 weeks and regeneration of spinal motoneurons and recovery of muscle weight evaluated at 16 weeks postoperatively. Schwann cells survived in the BD hydrogel both in culture and after transplantation into the nerve defect. Regenerating axons grew significantly longer distances within the conduits filled with BD hydrogel when compared with the alginate/fibronectin hydrogel and alginate/fibronectin with Schwann cells. Addition of Schwann cells to the BD hydrogel considerably increased regeneration distance with axons crossing the injury gap and entering into the distal nerve stump. The conduits with BD hydrogel showed a linear alignment of nerve fibers and Schwann cells. The number of regenerating motoneurons and recovery of the weight of the gastrocnemius muscle was inferior in BD hydrogel and alginate/fibronectin groups compared with nerve grafting. Addition of Schwann cells did not improve regeneration of motoneurons or muscle recovery. The present results suggest that BD hydrogel with Schwann cells could be used within biosynthetic conduits to increase the rate of axonal regeneration across a nerve defect.

Peripheral nerve injury induces the retrograde degeneration of dorsal root ganglion (DRG) cells, which affects predominantly the small-diameter cutaneous afferent neurons. This study compares the time-course of retrograde cell death in cutaneous and muscular DRG cells after peripheral nerve transection as well as neuronal survival and axonal regeneration after primary repair or nerve grafting. For comparison, spinal motoneurons were also included in the study. Sural and medial gastrocnemius DRG neurons were retrogradely labeled with the fluorescent tracers Fast Blue (FB) or Fluoro-Gold (FG) from the homonymous transected nerves. Survival of labeled sural and gastrocnemius DRG cells was assessed at 3 days and 1-24 weeks after axotomy. To evaluate axonal regeneration, the sciatic nerve was transected proximally at 1 week after FB-labeling of the sural and medial gastrocnemius nerves and immediately reconstructed using primary repair or autologous nerve grafting. Twelve weeks later, the fluorescent tracer Fluoro-Ruby (FR) was applied 10 mm distal to the sciatic lesion in order to double-label sural and gastrocnemius neurons that had regenerated across the repair site. Counts of labeled gastrocnemius DRG neurons did not reveal any significant retrograde cell death after nerve transection. In contrast, sural axotomy induced a delayed loss of sural DRG cells, which amounted to 22% at 4 weeks and 43-48% at 8-24 weeks postoperatively. Proximal transection of the sciatic nerve at 1 week after injury to the sural or gastrocnemius nerves neither further increased retrograde DRG degeneration, nor did it affect survival of sural or gastrocnemius motoneurons. Primary repair or peripheral nerve grafting supported regeneration of 53-60% of the spinal motoneurons and 47-49% of the muscular DRG neurons at 13 weeks postoperatively. In the cutaneous DRG neurons, primary repair or peripheral nerve grafting increased survival by 19-30% and promoted regeneration of 46-66% of the cells. The present results suggest that cutaneous DRG neurons are more sensitive to peripheral nerve injury than muscular DRG cells, but that their regenerative capacity does not differ from that of the latter cells. However, the retrograde loss of cutaneous DRG cells taking place despite immediate nerve repair would still limit the recovery of cutaneous sensory functions.

Motor recovery after proximal nerve injury remains extremely poor, despite advances in surgical care. Several neurobiological hurdles are implicated, the most fundamental being extensive cell death within the motorneuron pool. N-acetyl-cysteine almost completely protects sensory neurons after peripheral axotomy, hence its efficacy in protecting motorneurons after ventral root avulsion/rhizotomy was investigated. In adult rats, the motorneurons supplying medial gastrocnemius were unilaterally pre-labelled with retrograde tracer (true-blue/fluoro-gold), prior to L5 and 6 ventral root avulsion, or rhizotomy. Groups received either intraperitoneal N-acetyl-cysteine (ip, 150 or 750 mg/kg/day), immediate or delayed intrathecal N-acetyl-cysteine treatment (it, 2.4 mg/day), or saline; untreated animals served as controls. Either 4 (avulsion model) or 8 (rhizotomy model) weeks later, the pre-labelled motorneurons' mean soma area and survival were quantified. Untreated controls possessed markedly fewer motorneurons than normal due to cell death (avulsion 53% death; rhizotomy 26% death, P

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.

The optimal time for brachial plexus nerve repair is debatable. In this study we examined whether early re-establishment of neurotrophic support from the periphery might reduce neuronal loss. In 14 adult rats, the C7 spinal nerve was transsected. All sensory cells of the dorsal root ganglion and spinal motor neurons projecting into the C7 nerve were labelled retrogradely. The proximal and distal portions of the C7 nerve were then reanastomosed by either primary repair or by a vascularised or conventional ulnar nerve graft. At 16 weeks postoperatively, the nerve repair had significantly reduced the loss of both sensory and motor C7 neurons. Most striking was that a 30% motor neuronal loss in the control was almost eliminated by early nerve repair. In the grafted animals, half of the surviving neurons had regenerated through the graft, with no difference between vascularised and conventional nerve grafts. These results suggest that early surgical intervention may promote neuronal survival and regeneration after injuries to the brachial plexus.

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.

The existence of retrograde cell death in sensory dorsal root ganglion (DRG) cells after peripheral nerve injury is well established. However, with respect to retrograde motoneuron death after peripheral nerve injury, available data are conflicting. This may partly be due to the cell counting techniques used. In the present study, quantitative morphometric methods have been used to analyse retrograde motoneuron death induced by spinal nerve injury in adult rats. For comparison, DRG cells were also included in the study. The C7 spinal nerve was transected about 10 mm distal to the DRG and exposed to the fluorescent tracer fast blue in order to retrogradely label the spinal motoneurons and DRG cells of the C7 segment. At 1–16 weeks postoperatively, the nuclei of fast-blue-labelled C7 motoneurons and DRG cells were counted in consecutive 50-µm-thick serial sections. For comparison, the physical disector technique and measurements of neuronal density were also used to calculate motoneuron number. The counts of fast-blue-labelled motoneurons revealed a delayed motoneuron loss amounting to 21% and 31% after 8 and 16 weeks, respectively (P

The possibility of using the presence of the glial-cell-derived protein S-100 in serum as a marker for neuronal damage caused by spinal cord injury and plexus avulsion injury was investigated in 144 adult rats. After a spinal cord injury had been induced at the thoracic level or a plexus avulsion injury at the lumbar level, blood samples were taken and analysed for S-100 protein by a monoclonal two-site immunoluminometric assay. The two types of neurotrauma changed the kinetics of serum S-100 in different ways. After spinal cord injury it rapidly increased and within 72 hours had reached a concentration about 5 times that of the control animals. Three peak concentrations occurred at 3, 12, and 72 hours, respectively, and differed significantly from those of the control group (p < 0.05). After six days the values had returned to normal. After lumbar plexus injury alone there was no significant increase in the concentration of S-100. These results suggest that the concentration of S-100 protein in serum may be used as an early diagnostic tool for detecting neuronal damage caused by spinal cord injury or plexus avulsion associated with damage to the root entry zone.

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.

The effect of three different neurotrophins on axotomy-induced death of adult rat sensory neurons was examined. The ventral branch of the 13th spinal nerve was transected and the corresponding neurons in the 13th thoracic (T13) dorsal root ganglion (DRG) were pre-labelled with Fast Blue (FB). For a period of 4 weeks, animals received either no treatment, continuous intrathecal infusion of phosphate buffer, nerve growth factor (NGF), neurotrophin-3 (NT-3), or brain-derived neurotrophic factor (BDNF). Labelled neurons remaining after this period were counted. Inert, or no treatment, resulted in extensive loss of the DRG neurons. BDNF application was virtually non-effective, while NGF or NT-3 resulted in a greater number of FB-labelled neurons compared to normal controls. This suggests that NGF and NT-3 are survival factors for adult sensory neurons with a therapeutic potential in peripheral nerve injuries.

The permanence of retrograde neuronal labeling by the fluorescent tracers Fast Blue, Fluoro-Gold, Mini-Ruby, Fluoro-Ruby and Fluoro-Emerald was investigated in adult rat spinal motorneurons at 1, 4, 12 and 24 weeks after tracer application to a transected muscle nerve. After 1 week, the largest number of retrogradely labeled motoneurons was found with Mini-Ruby, Fluoro-Gold and Fluoro-Ruby, while Fluoro-Emerald yielded a smaller number of labeled cells. With increasing survival time, all of these tracers exhibited a marked decrease in the number of labeled neurons. Fast Blue also produced very efficient staining after 1 week and, in addition, the number of Fast Blue-labeled cells remained constant over the entire time period studied. Also in embryonic spinal cord tissue exposed to Fast Blue. the label persisted for at least 6 months after transplantation into adult spinal cord. Double-labeling experiments combining Fast Blue with Fluoro-Gold, Mini-Ruby, Fluoro-Ruby or Fluoro-Emerald showed that all these substances were non-toxic and that the time-related decrease in the number of neurons labeled by the latter tracers was due to degradation or leakage of the dyes. Thus, Fast Blue would be the tracer of choice for motoneuronal labeling in long-term experiments, whereas the usage of the other tracers should be restricted to experiments of limited duration.

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.

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.

Peripheral nerve injury leads to deficient recovery of sensation and a causative factor may be that only 50-60% of primary sensory neurons succeed in regenerating axons after primary nerve repair. In this study, an in vivo rat sciatic nerve injury and regeneration model was combined with laser microdissection and quantitative real-time polymerase chain reaction with the aim of examining the gene expression of regenerative molecules in cutaneous and muscular sensory neurons. Recent studies have identified peripherin and ATF-3 molecules as crucial for neurite outgrowth propagation; our novel findings demonstrate a subpopulation of non-regenerating sensory neurons characterized by a failure to upregulate transcription of these molecules and that a greater peripherin mRNA expression in injured cutaneous neurons may potentiate this subpopulation to regenerate more axons than muscle afferent neurons following injury. The gene expression of the structural neurofilament NF-H is found to be significantly downregulated following injury in both sensory subpopulations.

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.

Cavity formation is an important obstacle impeding regeneration after spinal cord injury and bridging strategies are essential to provide physical substrate allowing axons to grow across the lesion site. In this study we evaluated effects of biodegradable tubular conduit made from poly-beta-hydroxybutyrate (PHB) scaffold with predominantly unidirectional fiber orientation and supplemented with cultured adult Schwann cells on axonal regeneration after cervical spinal cord injury in adult rats. After transplantation into the injured spinal cord, plain PHB conduit was well-integrated into posttraumatic cavity and induced modest astroglial reaction. Regenerating axons were found mainly outside the PHB with only single fibers crossing the host-graft interface. No host Schwann cells migrated into the graft. In contrast, when suspension of adult Schwann cells was added to the PHB during transplantation, neurofilament-positive axons filled the conduit and became associated with the implanted cells. Although rubrospinal fibers did not enter the PHB, numerous raphaespinal and CGRP-positive axons were found within the conduit. Modification of PHB surface with fibronectin, laminin or collagen significantly increased Schwann cell attachment and proliferation in vitro. However, transplantation of PHB conduit pre-coated with fibronectin and seeded with Schwann cells did not alter axonal growth response. The results demonstrate that a PHB scaffold promotes attachment, proliferation and survival of adult Schwann cells and supports marked axonal regeneration within the graft.

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.

It has been proposed clinically that delayed surgery after traumatic brachial plexus injury may adversely affect functional outcome. In the present experimental study the neuroprotective and growth-promoting effects of early and delayed nerve grafting following proximal seventh cervical spinal nerve (C7) axotomy were examined. The ventral branch of C7 spinal nerve was transected and axons projecting out of the proximal nerve stump were labelled with Fast Blue (FB). At the same time, the biceps brachii muscle was denervated by transecting the musculocutaneous nerve at its origin. Neuronal survival and muscle atrophy were then assessed at 1, 4, 8 and 16 weeks after permanent axotomy. In the experimental groups, a peripheral nerve graft was interposed between the transected C7 spinal nerve and the distal stump of the musculocutaneous nerve at 1 week [early nerve repair (ENR)] or 8 weeks [delayed nerve repair (DNR)] after axotomy. Sixteen weeks after nerve repair had been performed, a second tracer Fluoro-Ruby (FR) was applied distal to the graft to assess the efficacy of axonal regeneration. Counts of FB-labelled neurons revealed that axotomy did not induce any significant cell loss at 4 weeks, but 15% of motoneurons and 32% of sensory neurons died at 8 weeks after injury. At 16 weeks, the amount of cell loss in spinal cord and dorsal root ganglion (DRG) reached 29 and 50%, respectively. Both ENR and DNR prevented retrograde degeneration of spinal motoneurons and counteracted muscle atrophy, but failed to rescue sensory neurons. Due to substantial cell loss at 8 weeks, the number of FR-labelled neurons after DNR was significantly lower when compared to ENR. However, the proportion of regenerating neurons among surviving motoneurons and DRG neurons remained relatively constant indicating that neurons retained their regenerative capacity after prolonged axotomy. The results demonstrate that DNR could protect spinal motoneurons and reduce muscle atrophy, but had little effect on sensory DRG neurons. However, the efficacy of neuroprotection and axonal regeneration will be significantly affected by the amount of cell loss already presented at the time of nerve repair.

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

The efficacy of anterograde labeling of the central projections of primary afferent fibers were compared between biotinylated dextran amine (BDA), neurobiotin (NB) and Phaseolus vulgaris-leucoagglutinin (PHA-L) after injections into the L5 or T13 dorsal root ganglia (DRGs) of adult rats. Excellent labeling was obtained with BDA, which visualized fibers with fine terminal boutons in the L5 and T13 spinal cord segments, Clarke's nucleus and the gracile nucleus. Rarely observed crossed projections to the gracile nucleus and L5 ventral horn of the contralateral side could also be distinguished. Even in the most successful experiments, however, BDA labeled only about one-third of the axons originating from the injected dorsal root ganglion. BDA was also efficient as transganglionic tracer after application to the transected sciatic nerve. NB produced no significant labeling of the L5 primary afferents, and was only moderately effective on the T13 level. PHA injections resulted in sparse terminal labeling of the T13 and L5 afferents. Thus, BDA is an effective tracer for long-range labeling of primary afferent projections in the spinal cord and brain stem. Since not all stem fibers become labeled, however, the method does not allow quantification of all axon branches and terminals arising from the injected DRGs.

Clinically optimal situations for primary nerve repair are rarely observed. Crushed nerve ends result in either suboptimal repair or a need for nerve grafting. Functional results after nerve surgery are relatively poor, including major sensory deficits, which may be due to the death of primary sensory neurons that follows the nerve injury. The aim of this study was to determine if using polyhydroxybutyrate (PHB), a resorbable nerve conduit, could be an alternative to primary nerve repair in reducing loss of neurons. The superficial radial nerves in 20 cats were sectioned bilaterally and primarily repaired microsurgically by the use of two different strategies ; either wrapping the nerve ends in sheets of PHB or epineurally suturing of the nerve. After 6 or 12 months, the surviving neurons within the dorsal root ganglia [C5-T1 ] were counted. No statistically significant differences were found between the two methods. This may imply a future possibility of using PHB as a synthetic nerve graft in situations where suboptimal primary repair or nerve grafts are the alternatives.

We compared the effects of peripheral nerve grafts, embryonic spinal cord transplants and brain-derived neurotrophic factor (BDNF) on the survival and axon regeneration of adult rat spinal motor neurons undergoing retrograde degeneration after ventral root avulsion. Following implantation into the dorsolateral funiculus of the injured spinal cord segment, neither a peripheral nerve graft nor a combination of peripheral nerve graft with embryonic spinal cord transplant could prevent the retrograde motor neuron degeneration induced by ventral root avulsion. However, intrathecal infusion of BDNF promoted long-term survival of the lesioned motor neurons and induced abundant motor axon regeneration from the avulsion zone along the spinal cord surface towards the BDNF source. A combination of ventral root reconstitution and BDNF treatment might therefore be a promising means for the support of both motor neuron survival and guided motor axon regeneration after ventral root lesions.

Spinal cord injury (SCI) often results in necrotic changes leading to cavity formation and glial scar tissue in the lesion zone. We have examined the effects of continuous topical administration of brain-derived neurotrophic factor (BDNF) on cavity formation and neuronal death after SCI. Following retrograde prelabeling of the tibial motoneurons in the L4-L6 spinal cord segments with the fluorescent dye Fast blue, a spinal hemisection was performed in the L5 segment. At 4 weeks postoperatively, only 66% of the labeled motoneurons remained in the untreated animals, while BDNF treatment resulted in a significant reduction in size of the lesion cavity and 92% motoneuron survival. A therapeutic potential of BDNF in the early treatment of SCI is suggested.

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.