BDNF production by olfactory ensheathing cells contributes to axonal regeneration of cultured adult CNS neurons

Olfactory ensheathing cells (OECs) are the main glial cell type that populates mammalian olfactory nerves. These cells have a great capacity to promote the regeneration of axons when transplanted into the injured adult mammalian CNS. However, little is still known about the molecular mechanisms they employ in mediating such a task. Brain-derived neurotrophic factor (BDNF) was identified as a candidate molecule in a genomic study that compared three functionally different OEC populations: Early passage OECs (OEC Ep), Late passage OECs (OEC Lp) and the OEC cell line TEG3 [Pastrana, E., Moreno-Flores, M.T., Gurzov, E.N., Avila, J., Wandosell, F., Diaz-Nido, J., 2006. Genes associated with adult axon regeneration promoted by olfactory ensheathing cells: a new role for matrix metalloproteinase 2. J. Neurosci. 26, 5347-5359]. We have here set out to determine the role played by BDNF in the stimulation of axon outgrowth by OECs. We compared the extracellular BDNF levels in the three OEC populations and show that it is produced in significant amounts by the OECs that can stimulate axon regeneration in adult retinal neurons (OEC Ep and TEG3) but it is absent from the extracellular medium of OEC Lp cells which lack this capacity. Blocking BDNF signalling impaired axonal regeneration of adult retinal neurons co-cultured with TEG3 cells and adding BDNF increased the proportion of adult neurons that regenerate their axons on OEC Lp monolayers. Combining BDNF with other extracellular proteins such as Matrix Metalloproteinase 2 (MMP2) further augmented this effect. This study shows that BDNF production by OECs plays a direct role in the promotion of axon regeneration of adult CNS neurons.     

Olfactory ensheathing cells: their role in central nervous system repair

The olfactory system is an unusual tissue in that it can support neurogenesis throughout life; permitting the in-growth and synapse formation of olfactory receptor axons into the central nervous system (CNS) environment of the olfactory bulb. It is thought that this unusual property is in part due to the olfactory glial cells, termed olfactory ensheathing cells (OECs), but also due to neuronal stem cells. These glial cells originate from the olfactory placode and possess many properties in common with the glial cells from the peripheral nervous system (PNS), Schwann cells. Recent data has suggested that olfactory ensheathing cells are a distinct glial cell type and possess properties, which might make them more suitable for transplant-mediated repair of central nervous system injury models. This paper reviews the biological properties of these cells and illustrates their use in central nervous system repair.     

Neutralization of BDNF Attenuates the in vitro Protective Effects of Olfactory Ensheathing Cell-Conditioned Medium on Scratch-Insulted Retinal Ganglion Cells

Transplantation of olfactory ensheathing cells (OECs) becomes one of the promising strategies in restoring lost functions of injured central nervous system. Elevated level of expressed brain-derived neurotrophic factor (BDNF) was revealed in the previous studies to be related to the protective effects of OECs on injured cortical and brain stem neurons as well as retinal ganglion cells (RGCs), but no evidence has been obtained to demonstrate whether transplanted OECs protect injured central neurons directly by their secreted BDNF. In the present study, the effects of BDNF neutralization on the neuroprotection of adult OEC-conditioned medium (OEC-CM) on scratch-insulted RGCs were examined. The results showed that OEC-CM protected cultured RGCs from scratch insult, and neutralization of BDNF by BDNF neutralizing antibody attenuated such neuroprotection of the medium. It is thus concluded that neurotrophic factors including BDNF secreted by OECs can protect injured OECs in vitro and BDNF plays a major role in such a protection of OECs.     

Molecular mechanisms for migration of placodally derived GnRH neurons

Gonadotropin-releasing hormone (GnRH) neurons, critical for reproduction, are derived from the nasal placode and migrate into the brain along nasal axons. GnRH neurons appear to diverge from olfactory sensory cells during early stages of nasal placode differentiation. However, GnRH neurons rely on olfactory/vomeronasal axons as their pathway to the central nervous system (CNS). A novel factor, termed nasal embryonic luteinizing hormone-releasing hormone factor (NELF), was discovered in a differential screen of migrating versus nonmigrating GnRH neurons. NELF is expressed in olfactory sensory cells and GnRH cells in nasal areas. Antisense experiments demonstrated that knock-down of NELF decreased olfactory axon outgrowth and GnRH neuronal migration. These results indicate that NELF plays a role as a guidance molecule for olfactory axon projections and migration of GnRH cells. We hypothesize that NELF acts via a homophilic interaction and that NELF expression is critical for reproduction by insuring that GnRH cells reach the CNS. Furthermore, down-regulation of NELF on GnRH cells as they enter the telencephalon may allow GnRH cells to distinguish a different pathway(s) in the CNS (from those leading to olfactory regions) and thereby facilitate establishment of the appropriate adult-like GnRH distribution.     

Phagocytic Removal of Neuronal Debris by Olfactory Ensheathing Cells Enhances Neuronal Survival and Neurite Outgrowth via p38MAPK Activity

Compelling evidence from animal models and clinical studies suggest that transplantation of olfactory ensheathing cells (OECs), specialized glia in the olfactory system, combined with specific training may be therapeutically useful in the central nervous system (CNS) injuries and neurodegenerative diseases. The unique function of OECs could mainly attribute to both production of cell adhesion molecules and secretion of growth factors in OECs, which support neuron survival and neurite outgrowth. However, little is known about whether engulfment of neuronal degenerative debris by OECs also equally contributes to neuronal survival and neurite outgrowth. Furthermore, the molecular mechanisms responsible for neuronal degenerative corpses' removal remain elusive. Here, we used an in vitro model of primary culture of spinal cord neurons to investigate the effect of engulfment of degenerative neuron debris by OECs on neuronal survival and neurite outgrowth and the possible molecular mechanisms. Our results showed that OECs can engulf an amount of degenerated neuron debris, and this phagocytosis can make a substantial contribution to neuron growth, as demonstrated by increased number of neurons with longer neurite length and richer neurite branches when compared with the combination of neuron debris and OEC conditioned medium (OECCM). Moreover, p38 mitogen-activated protein kinase (p38MAPK) signaling pathway may mediate the OEC engulfment of debris because the p38MAPK-specific inhibitor, SB203580, can abrogate all the positive effects of OECs, including clearance of degenerated neuron debris and generation of bioactive molecules, indicating that p38MAPK is required for the process of phagocytosis of the neuron debris. In addition, the OEC phagocytic activity had no influence on its generation of bioactive molecules. Therefore, these findings provide new insight into further investigations on the OEC role in the repair of traumatic CNS injury and neurodegenerative diseases.     

Strategies to repair lost sensory connections to the spinal cord

Failure of injured axons to regenerate in the central nervous system (CNS) is the main obstacle for repair of stroke and traumatic injuries to the spinal cord and sensory roots. This regeneration failure is high-lighted at the dorsal root transitional zone (DRTZ), the boundary between the peripheral (PNS) and central nervous system where sensory axons enter the spinal cord. Injured sensory axons regenerate in the PNS compartment of the dorsal root but are halted as soon as they reach the DRTZ. The failure of regenerating dorsal root axons to re-enter the mature spinal cord is a reflection of the generally nonpermissive nature of the CNS environment, in contrast to the regeneration supportive properties of the PNS. The dorsal root injury paradigm is therefore an attractive model for studying mechanisms underlying CNS regeneration failure in general and how to overcome the hostile CNS environment. Here we review the main lines that have been pursued to achieve growth of injured dorsal root axons into the spinal cord: (i) modifying the inhibitory nature of the DRTZ by breaking down or blocking the effect of growth repelling molecules, (ii) stimulate elongation of injured dorsal root axons by a prior conditioning lesion or administration of specific growth factors, (iii) implantation of olfactory ensheathing cells to provide a growth supportive cellular terrain at the DRTZ, and (iv) replacing the regeneration deficient adult dorsal root ganglion neurons with embryonic neurons or neural stem cells.     

The migration of olfactory ensheathing cells during development and regeneration

The primary olfactory nervous system is unique in that it continuously renews itself and regenerates after injury. These properties are attributed to the presence of olfactory glia, termed olfactory ensheathing cells (OECs). Evidence is now emerging that individual OEC populations exist with distinct anatomical localisations and physiological properties, but their differential roles have not been determined. Unlike other glia, OECs can migrate from the periphery into the central nervous system, and organised OEC migration can enhance axonal extension after injury. Despite this, the mechanisms regulating OEC migration are largely unknown. Here, we provide an overview of the roles of OECs in development and adulthood. We review the latest research describing the differences between individual OEC subpopulations and discuss potential regulatory mechanisms for OEC guidance and migration. Using advanced time lapse techniques, we have obtained novel insights into how OECs behave in a complex multicellular environment which we discuss here with particular focus on cell-cell interactions. Significantly, transplantation of OECs constitutes a promising novel therapy for nerve injuries, but results are highly variable and the method needs improvement. We here review the roles of transplanted OECs in neural repair of damaged neuronal tracts distinct from the primary olfactory nervous system.     

Central Nervous System Lesions That Can and Those That Cannot Be Repaired with the Help of Olfactory Bulb Ensheathing Cell Transplants

Growth-promoting macroglia (aldynoglia) with growth properties and immunological markers similar to Schwann cells, are found in loci of the mammalian CNS where axon regeneration occurs throughout life, like the olfactory sytem, hypothalamus-hypophysis and the pineal gland [79]. Contrary to Schwann cells, aldynoglia mingle freely with astrocytes and can migrate in brain and spinal cord. Transplantation of cultured and immunopurified olfactory ensheathing cells (OECs) in the spinal cord after multiple central rhizotomy, promoted sensory and central axon growth and partial functional restoration, judging by anatomical, electrophysiological and behavioural criteria. OEC transplants suppressed astrocyte reactivity, thus generally favouring axon growth after a lesion. However, the functional repair promoted by OEC transplants was partial in the best cases, depending on lesion type and location. Cyst formation after photochemical cord lesion was partially prevented but neither the corticospinal tract, interrupted by a mild contusion, nor the sectioned medial longitudinal fascicle, did regrow after OEC transplantation in the injured area.     

Fibroblasts Isolated from Human Middle Turbinate Mucosa Cause Neural Progenitor Cells to Differentiate into Glial Lineage Cells

Transplantation of olfactory ensheathing cells (OECs) is a potential therapy for repair of spinal cord injury (SCI). Autologous transplantation of OECs has been reported in clinical trials. However, it is still controversial whether purified OECs or olfactory mucosa containing OECs, fibroblasts and other cells should be used for transplantation. OECs and fibroblasts were isolated from olfactory mucosa of the middle turbinate from seven patients. The percentage of OECs with p75^NTR+ and GFAP⁺ ranged from 9.2% to 73.2%. Fibroblasts were purified and co-cultured with normal human neural progenitors (NHNPs). Based on immunocytochemical labeling, NHNPs were induced into glial lineage cells when they were co-cultured with the mucosal fibroblasts. These results demonstrate that OECs can be isolated from the mucosa of the middle turbinate bone as well as from the dorsal nasal septum and superior turbinates, which are the typical sites for harvesting OECs. Transplantation of olfactory mucosa containing fibroblasts into the central nervous system (CNS) needs to be further investigated before translation to clinical application.     

RhoA-ROCK-Myosin pathway regulates morphological plasticity of cultured olfactory ensheathing cells

Olfactory ensheathing cells (OECs) are glial cells in the olfactory system with morphological and functional plasticity. Cultured OECs have the flattened and process-bearing shape. Reversible changes have been found between these two morphological phenotypes. However, the molecular mechanism underlying the regulation of their morphological plasticity remains elusive. Using RhoA FRET biosensor, we found that the active RhoA signal mainly distributed in the lamellipodia and/or filopodia of OECs. Local disruption of these active RhoA distributions led to the morphological change from the flattened into process-bearing shape and promoted process outgrowth. Furthermore, RhoA pathway inhibitors, Toxin-B, C3, Y-27632 or over-expression of DN-RhoA blocked serum-induced morphological change of OECs from the process-bearing into flattened shape, whereas the activation of RhoA pathway by lysophosphatidic acid (LPA) promoted the morphological change from the process-bearing into flattened shape. Finally, ROCK–Myosin–F-actin as a downstream of RhoA pathway was involved in morphological plasticity of OECs. Taken together, these results suggest that RhoA–ROCK–Myosin pathway mediates the morphological plasticity of cultured OECs in response to extracellular cues.     

Co-transplantation of olfactory ensheathing cells from mucosa and bulb origin enhances functional recovery after peripheral nerve lesion

Olfactory ensheathing cells (OECs) represent an interesting candidate for cell therapy and could be obtained from olfactory mucosa (OM-OECs) or olfactory bulbs (OB-OECs). Recent reports suggest that, depending on their origin, OECs display different functional properties. We show here the complementary and additive effects of co-transplanting OM-OECs and OB-OECs after lesion of a peripheral nerve. For this, a selective motor denervation of the laryngeal muscles was performed by a section/anastomosis of the recurrent laryngeal nerve (RLN). Two months after surgery, recovery of the laryngeal movements and synkinesis phenonema were analyzed by videolaryngoscopy. To complete these assessments, measure of latency and potential duration were determined by electrophysiological recordings and myelinated nerve fiber profiles were defined based on toluidine blue staining. To explain some of the mechanisms involved, tracking of GFP positive OECs was performed. It appears that transplantation of OM-OECs or OB-OECs displayed opposite abilities to improve functional recovery. Indeed, OM-OECs increased recuperation of laryngeal muscles activities without appropriate functional recovery. In contrast, OB-OECs induced some functional recovery by enhancing axonal regrowth. Importantly, co-transplantation of OM-OECs and OB-OECs supported a major functional recovery, with reduction of synkinesis phenomena. This study is the first which clearly demonstrates the complementary and additive properties of OECs obtained from olfactory mucosa and olfactory bulb to improve functional recovery after transplantation in a nerve lesion model.     

Expression of extracellular matrix molecules in the embryonic rat olfactory pathway

Primary olfactory neurons arise from placodal neuroepithelium that is separate from the neuroepithelial plate that forms the neural tube and crest. The axons of these neurons course along a stereotypical pathway and invade the rostral telencephalic vesicle where they induce the formation of the olfactory bulb. In the present study we examined the expression of several extracellular matrix constituents during formation of the olfactory nerve pathway in order to identify putative developmentally significant molecules. Double-label immunofluorescence was used to simultaneously map the trajectory of growing primary olfactory axons by expression of growth associated protein 43 (GAP-43) and the distribution of either laminin, heparan sulfate proteoglycans (HSPG), or chondroitin sulfate proteoglycans (CSPG). At embryonic day 12.5 (E12.5) primary olfactory axons have exited the olfactory neuroepithelium of the nasal pit and formed a rudimentary olfactory nerve. These axons together with migrating neural cells form a large mass outside the rostral surface of the telencephalon. This nerve pathway is clearly defined by a punctate distribution of laminin and HSPG. CSPG is selectively present in the mesenchyme between the olfactory nerve pathway and the nasal pit and in the marginal zone of the telencephalon. At E14.5 primary olfactory axons pierce the telencephalon through gaps that have emerged in the basement membrane. At this age both laminin and HSPG are colocalized with the primary olfactory axons that have entered the marginal zone of the telencephalon. CSPG expression becomes downregulated in this same region while it remains highly expressed in the marginal zone adjacent to the presumptive olfactory bulb. By E16.5 most of the basement membrane separating the olfactory nerve from the telencephalon has degraded, and there is direct continuity between the olfactory nerve pathway and the central nervous system. This strict spatiotemporal regulation of extracellular matrix constituents in the olfactory nerve pathway supports an important role of these molecules in axon guidance. We propose that laminin and HSPG are expressed by migrating olfactory Schwann cells in the developing olfactory nerve pathway and that these molecules provide a conducive substrate for axon growth between the olfactory neuroepithelium and the brain. CSPG in the surrounding mesenchyme may act to restrict axon growth to within this pathway. The regional degradation of the basement membrane of the telencephalon and the downregulation of CSPG within the marginal zone probably facilitates the passage of primary olfactory axons into the brain to form the presumptive nerve fiber layer of the olfactory bulb. © 1996 John Wiley & Sons, Inc.     

Molecular composition of tight and adherens junctions in the rat olfactory epithelium and fila

Tight and adherens junctions (TJs, AJs) between neurons, epithelial and glial cells provide barrier and adhesion properties in the olfactory epithelium (OE), and subserve functions such as compartmentalization and axon growth in the fila olfactoria (FO). Immunofluorescence and immunoelectronmicroscopy were combined in sections of rat OE and FO to document the cellular and subcellular localization of TJ proteins occludin(Occl), claudins(Cl) 1-5 and zonula occludens(ZO) proteins 1-3, and of AJ proteins N-cadherin(cad), E-cad, and alpha-, beta- and p120-catenin(cat). With the exception of Cl2, all TJ proteins were colocalized in OE junctions. Differences in relative immunolabeling intensities were noted between neuronal and epithelial TJs. In the FO, Cl5-reactivity was localized in olfactory ensheathing cell (OEC) junctions, Cl1-reactivity in the FO periphery, with differential colocalization with ZOs. Supporting cells formed N-cad-immunoreactive (ir) AJs with olfactory sensory neurons, E-cad-ir junctions with microvillar and gland duct cells, and both N-cad and E-cad-ir junctions in homotypic contacts. Alpha, beta- and p120-cat were localized in all AJs of the OE. AJs were scarce in the globose basal cell layer. Immature and mature neurons formed numerous contacts. In the FO, AJs were documented between OECs, between OECs and axons, and between axons. Most AJs colocalized N-cad with catenins, occasionally E-cad-ir AJs were found in the FO periphery. Characteristics of molecular composition suggest differential properties of TJs formed by neuronal, epithelial and glial cells in the OE and FO. The presence and molecular composition of AJs are consistent with a role of AJ proteins in neuroplastic processes in the peripheral olfactory pathway.     

Reactive Astrocytes in Glial Scar Attract Olfactory Ensheathing Cells Migration by Secreted TNF-α in Spinal Cord Lesion of Rat

Background

After spinal cord injury (SCI), the formation of glial scar contributes to the failure of injured adult axons to regenerate past the lesion. Increasing evidence indicates that olfactory ensheathing cells (OECs) implanted into spinal cord are found to migrate into the lesion site and induce axons regeneration beyond glial scar and resumption of functions. However, little is known about the mechanisms of OECs migrating from injection site to glial scar/lesion site.

Methods and Findings

In the present study, we identified a link between OECs migration and reactive astrocytes in glial scar that was mediated by the tumor necrosis factor-α (TNF-α). Initially, the Boyden chamber migration assay showed that both glial scar tissue and reactive astrocyte-conditioned medium promoted OECs migration in vitro. Reactive astrocyte-derived TNF-α and its type 1 receptor TNFR1 expressed on OECs were identified to be responsible for the promoting effect on OECs migration. TNF-α-induced OECs migration was demonstrated depending on activation of the extracellular signal-regulated kinase (ERK) signaling cascades. Furthermore, TNF-α secreted by reactive astrocytes in glial scar was also showed to attract OECs migration in a spinal cord hemisection injury model of rat.

Conclusions

These findings showed that TNF-α was released by reactive astrocytes in glial scar and attracted OECs migration by interacting with TNFR1 expressed on OECs via regulation of ERK signaling. This migration-attracting effect of reactive astrocytes on OECs may suggest a mechanism for guiding OECs migration into glial scar, which is crucial for OECs-mediated axons regrowth beyond the spinal cord lesion site.     

Remyelination of spinal cord axons by olfactory ensheathing cells and Schwann cells derived from a transgenic rat expressing alkaline phosphatase marker gene

Transplantation of cell suspensions containing olfactory ensheathing cells (OECs) has been reported to remyelinate demyelinated axons in the spinal cord with a Schwann cell (SC)-like pattern of myelination. However, questions have been raised recently as to whether OECs can form SC-like myelin. To address this issue we prepared SCs and OECs from transgenic rats in which a marker gene, human placental alkaline phosphatase (hPAP), is linked to the ubiquitously active promoter of the R26 gene. SCs were prepared from the sciatic nerve and OECs from the outer nerve-fiber layer of the olfactory bulb. Positive S100 and p75 immunostaining indicated that >95% of cells in culture displayed either SC or OEC phenotypes. Suspensions of either SCs or OECs were transplanted into an X-irradiation/ethidium bromide demyelinating lesion in the spinal cord. We observed extensive SC-like remyelination following either SC or OEC transplantation 3 weeks after injection of the cells. Alkaline phosphatase (ALP) chromagen reaction product was associated clearly with the myelin-forming cells. Thus, cell suspensions that are enriched in either SCs or OECs result in peripheral-like myelin when transplanted in vivo.     

Transplantation of olfactory ensheathing cells fails to promote significant axonal regeneration from dorsal roots into the rat cervical cord

The olfactory ensheathing cell (OEC) is a class of glial cell that has been reported to support regeneration in the central nervous system after various types of lesions, including rhizotomy of spinal dorsal roots at thoracic, lumbar and sacral levels. We have therefore carried out a detailed anatomical analysis to assess the efficacy of dorsal horn OEC transplants at promoting regeneration of primary afferents across the dorsal root entry zone (DREZ) at the cervical level in the adult rat. OECs were cultured from adult rat olfactory bulb and immunopurified (90% purity). Regeneration by large diameter afferents and by both peptidergic and non-peptidergic small diameter afferents was assessed using respectively cholera toxin B (CTB) labelling and immunocytochemistry for calcitonin gene-related peptide (CGRP) and the purinoceptor P2X3. Following an extensive (C3-T3) rhizotomy, CGRP and P2X3 immunoreactive axons regenerated across the rhizotomy site as far as the DREZ but there was no evidence of regeneration across the DREZ, except through sites where the OEC transplant was directly grafted into the DREZ. No evidence of regeneration into the dorsal horn by CTB-labelled axons was obtained. In addition, there was little sign of sprouting by intact axons in the vicinity of OEC transplant sites. In contrast to these results in vivo, cocultures of OECs and adult dorsal root ganglion cells showed that OECs stimulate extensive neurite outgrowth. The failure of the OECs to promote regeneration in vivo following cervical rhizotomy is therefore most likely due to factors in the environment of the graft site and/or the method of transplantation.     

Implications of Olfactory Lamina Propria Transplantation on Hyperreflexia and Myelinated Fiber Regeneration in Rats with Complete Spinal Cord Transection

Transplantation with olfactory ensheathing cells (OECs) has been adopted after several models of spinal cord injury (SCI) with the purpose of creating a favorable environment for the re-growth of injured axons. However, a consensus on the efficacy of this cellular transplantation has yet to be reached. In order to explore alternative parameters that could demonstrate the possible restorative properties of such grafts, the present study investigated the effects of olfactory lamina propria (OLP) transplantation on hyperreflexia and myelinated fiber regeneration in adult rats with complete spinal cord transection. The efficacy of OLP (graft containing OECs) and respiratory lamina propria (RLP, graft without OECs) was tested at different post-injury times (acutely, 2- and 4-week delayed), to establish the optimum period for transplantation. In the therapeutic windows used, OLP and RLP grafts produced no considerable improvements in withdrawal reflex responses or on the low-frequency dependent depression of H-reflex. Both lamina propria grafts produced comparable results for the myelinated fiber density and for the estimated total number of myelinated fibers at the lesion site, indicating that the delayed transplantation approach does not seem to limit the regenerative effects. However, animals transplanted with OLP 2 or 4 weeks after injury exhibit smaller myelin sheath thickness and myelinated fiber area and diameter at the lesion site compared to their respective RLP groups. Despite the ongoing clinical use of OECs, it is important to emphasize the need for more experimental studies to clarify the exact nature of the repair capacity of these grafts in the treatment of SCI.     

Soluble Axoplasm Enriched from Injured CNS Axons Reveals the Early Modulation of the Actin Cytoskeleton

Axon injury and degeneration is a common consequence of diverse neurological conditions including multiple sclerosis, traumatic brain injury and spinal cord injury. The molecular events underlying axon degeneration are poorly understood. We have developed a novel method to enrich for axoplasm from rodent optic nerve and characterised the early events in Wallerian degeneration using an unbiased proteomics screen. Our detergent-free method draws axoplasm into a dehydrated hydrogel of the polymer poly(2-hydroxyethyl methacrylate), which is then recovered using centrifugation. This technique is able to recover axonal proteins and significantly deplete glial contamination as confirmed by immunoblotting. We have used iTRAQ to compare axoplasm-enriched samples from naïve vs injured optic nerves, which has revealed a pronounced modulation of proteins associated with the actin cytoskeleton. To confirm the modulation of the actin cytoskeleton in injured axons we focused on the RhoA pathway. Western blotting revealed an augmentation of RhoA and phosphorylated cofilin in axoplasm-enriched samples from injured optic nerve. To investigate the localisation of these components of the RhoA pathway in injured axons we transected axons of primary hippocampal neurons in vitro. We observed an early modulation of filamentous actin with a concomitant redistribution of phosphorylated cofilin in injured axons. At later time-points, RhoA is found to accumulate in axonal swellings and also colocalises with filamentous actin. The actin cytoskeleton is a known sensor of cell viability across multiple eukaryotes, and our results suggest a similar role for the actin cytoskeleton following axon injury. In agreement with other reports, our data also highlights the role of the RhoA pathway in axon degeneration. These findings highlight a previously unexplored area of axon biology, which may open novel avenues to prevent axon degeneration. Our method for isolating CNS axoplasm also represents a new tool to study axon biology.     

Cell surface expression of 27C7 by neonatal rat olfactory ensheathing cells in situ and in vitro is independent of axonal contact

Olfactory ensheathing cells (OECs) are Schwann cell-like glial cells of the olfactory system that promote neural regeneration after transplantation into the injured central nervous system. Compared to the closely related Schwann cells, however, the biological characterization of OECs has remained fragmentary. This is due to the fact that the expression of OEC-specific markers is subject to complex regulation and that intricate ultrastructural analysis is essential to determine their localization. The p75 neurotrophin receptor (p75^NTR) as the prototype OEC marker, for example, is only expressed by a minor population of neonatal rat OECs in situ. The major population carries O4-positive axonal fragments on their surface after dissociation and up-regulates p75^NTR during culturing (Wewetzer et al. in Glia 49:577–587, 2005). In the present study, we investigated whether the cell surface determinant 27C7, defined by a monoclonal antibody to Schwann cells, is also expressed by neonatal rat OECs in situ and in vitro. Primary cell suspensions of the olfactory bulb displayed 27C7 expression of both p75^NTR-negative and p75^NTR-positive OECs, while immature oligodendrocytes and astrocytes were devoid of any 27C7 labeling. This together with the finding that the intrafascicular OECs of the olfactory nerves in the mucosa expressed 27C7 but not p75^NTR, suggests that 27C7 was expressed by the entire OEC population in situ. Maintenance of OECs in the absence of olfactory neurons in organotypic slice culture up-regulated p75^NTR but did not alter 27C7 expression. It is concluded that 27C7 unlike p75^NTR is constitutively expressed by OECs and may, therefore, be a useful marker for characterization of neonatal OECs in situ and in vitro.