Human Embryonic Stem Cell-derived Neural Crest Cells Promote Sprouting and Motor Recovery Following Spinal Cord Injury in Adult Rats.

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.

Evaluation of apoptotic pathways in dorsal root ganglion neurons following peripheral nerve injury.

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.

Regenerative effects of human embryonic stem cell-derived neural crest cells for treatment of peripheral 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.

Trimethylene carbonate-caprolactone conduit with poly-p-dioxanone microfilaments to promote regeneration after spinal cord injury.

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. STATEMENT OF SIGNIFICANCE: 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.

Long-Term Effects of Fibrin Conduit with Human Mesenchymal Stem Cells and Immunosuppression after Peripheral Nerve Repair in a Xenogenic Model.

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.

A Morphological and Molecular Characterization of the Spinal Cord after Ventral Root Avulsion or Distal Peripheral Nerve Axotomy Injuries in Adult Rats.

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.

Differentiation of Pre- and Postganglionic Nerve Injury Using MRI of the Spinal Cord.

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 postganglionic, 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 magnetic resonance imaging (MRI) of the spinal cord for determination of injury level. The findings 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 postganglionic 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-neurofilament 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.

Chitosan polyplex mediated delivery of miRNA-124 reduces activation of microglial cells in vitro and in rat models 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 72 h 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 EDITOR: The 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.

Investigation of the Expression of Myogenic Transcription Factors, microRNAs and Muscle-Specific E3 Ubiquitin Ligases in the Medial Gastrocnemius and Soleus Muscles 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.

The therapeutic effects of human adipose-derived stem cells in a rat cervical spinal cord injury model.

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.

Stimulating the neurotrophic and angiogenic properties of human adipose-derived stem cells enhances nerve repair.

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.

Neuronal myosin-X is upregulated after peripheral nerve injury and mediates laminin-induced growth of neurites.

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.

Effect of delayed peripheral nerve repair on nerve regeneration, Schwann cell function and target muscle recovery.

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.

Aging effect on neurotrophic activity of human mesenchymal stem cells.

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.

Neuroprotective effects of N-acetyl-cysteine and acetyl-L-carnitine after spinal cord injury in adult rats.

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.

Fibrin conduit supplemented with human mesenchymal stem cells and immunosuppressive treatment enhances regeneration after peripheral nerve injury.

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.

Muscle recovery after repair of short and long peripheral nerve gaps using fibrin conduits.

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 (P<0.01) 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.

Neuroprotective and growth-promoting effects of bone marrow stromal cells after cervical spinal cord injury in adult rats.

BACKGROUND AIMS: Bone marrow stromal cells (BMSC) have been shown to provide neuroprotection after transplantation into the injured central nervous system. The present study investigated whether adult rat BMSC differentiated along a Schwann cell lineage could increase production of trophic factors and support neuronal survival and axonal regeneration after transplantation into the injured spinal cord. METHODS: After cervical C4 hemi-section, 5-bromo-2-deoxyuridine (BrdU)/green fluorescent protein (GFP)-labeled BMSC were injected into the lateral funiculus at 1 mm rostral and caudal to the lesion site. Spinal cords were analyzed 2-13 weeks after transplantation. RESULTS AND CONCLUSIONS: Treatment of native BMSC with Schwann cell-differentiating factors significantly increased production of brain-derived neurotrophic factor in vitro. Transplanted undifferentiated and differentiated BMSC remained at the injection sites, and in the trauma zone were often associated with neurofilament-positive fibers and increased levels of vascular endothelial growth factor. BMSC promoted extensive in-growth of serotonin-positive raphaespinal axons and calcitonin gene-related peptide (CGRP)-positive dorsal root sensory axons into the trauma zone, and significantly attenuated astroglial and microglial cell reactions, but induced aberrant sprouting of CGRP-immunoreactive axons in Rexed's lamina III. Differentiated BMSC provided neuroprotection for axotomized rubrospinal neurons and increased the density of rubrospinal axons in the dorsolateral funiculus rostral to the injury site. The present results suggest that BMSC induced along the Schwann cell lineage increase expression of trophic factors and have neuroprotective and growth-promoting effects after spinal cord injury.

Efficacy of olfactory ensheathing cells to support regeneration after spinal cord injury is influenced by method of culture preparation.

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.

BD™ PuraMatrix™ peptide hydrogel seeded with Schwann cells for peripheral nerve regeneration.

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.