Olfactory ensheathing cells (OECs) are non-myelinating glial cells that provide ensheathment for axons of the olfactory nerve in vivo. OECs have been shown to facilitate the regeneration of CNS axons, to assemble a myelin sheath around demyelinated axons, and it has been suggested OECs migrate very well within the microenvironment of the injured CNS. However, there has been no direct test of their migratory ability in vivo. The aims of this study were to determine whether: 1) OECs can be induced to migrate towards an ethidium bromide (EtBr)-induced focal (~1 mm long) demyelination of the spinal cord white matter; 2) OECs migrate away from a focal demyelination either into normal CNS tissue or towards a second demyelinated lesion; 3) microglial reactivity is required for the generation of the migratory signal(s) inducing OECs to migrate towards a focal demyelination; 4) OECs grafted into the subarachnoid space surrounding the spinal cord will migrate into the neuropil in the absence of demyelination. To achieve these aims, we developed an in vivo model for studying the migratory ability of OECs within the adult rat spinal cord. A small focal EtBr-induced demyelination of the dorsal funiculus (unilaterally) of the spinal cord was made at variable distances from the site of a DiI-labelled OEC graft. The major findings were: i) the strength of the migratory signal(s) inducing OECs to migrate increased as the demyelinated lesion was located closer to the grafting site; ii) the OEC migration towards a distal demyelinated lesion was greatly enhanced when the cells were grafted directly into a second demyelinated lesion; iii) the cell migration occurred along a migratory path containing many reactive astrocytes and microglia; iv) the migration of OECs was significantly reduced when the microglial reactivity was dampened using minocycline; and v) OECs survived grafting into cerebrospinal fluid (i.e. subarachnoid space) and migrated into the neuropil of the brain and spinal cord. The major conclusions are that OECs can respond to migratory signal(s) arising as a result of a focal EtBr-induced demyelination and that microglia are one potential source of these migratory signal(s).

Olfactory ensheathing cells (OECs) are non-myelinating glial cells that provide ensheathment for axons of the olfactory nerve in vivo. OECs have been shown to facilitate the regeneration of CNS axons, to assemble a myelin sheath around demyelinated axons, and it has been suggested OECs migrate very well within the microenvironment of the injured CNS. However, there has been no direct test of their migratory ability in vivo. The aims of this study were to determine whether: 1) OECs can be induced to migrate towards an ethidium bromide (EtBr)-induced focal (~1 mm long) demyelination of the spinal cord white matter; 2) OECs migrate away from a focal demyelination either into normal CNS tissue or towards a second demyelinated lesion; 3) microglial reactivity is required for the generation of the migratory signal(s) inducing OECs to migrate towards a focal demyelination; 4) OECs grafted into the subarachnoid space surrounding the spinal cord will migrate into the neuropil in the absence of demyelination. To achieve these aims, we developed an in vivo model for studying the migratory ability of OECs within the adult rat spinal cord. A small focal EtBr-induced demyelination of the dorsal funiculus (unilaterally) of the spinal cord was made at variable distances from the site of a DiI-labelled OEC graft. The major findings were: i) the strength of the migratory signal(s) inducing OECs to migrate increased as the demyelinated lesion was located closer to the grafting site; ii) the OEC migration towards a distal demyelinated lesion was greatly enhanced when the cells were grafted directly into a second demyelinated lesion; iii) the cell migration occurred along a migratory path containing many reactive astrocytes and microglia; iv) the migration of OECs was significantly reduced when the microglial reactivity was dampened using minocycline; and v) OECs survived grafting into cerebrospinal fluid (i.e. subarachnoid space) and migrated into the neuropil of the brain and spinal cord. The major conclusions are that OECs can respond to migratory signal(s) arising as a result of a focal EtBr-induced demyelination and that microglia are one potential source of these migratory signal(s).

Olfactory ensheathing cells (OECs) are glial cells that are an attractive candidate for neural repair after spinal cord injury and for remyelination of axons in diseases such as multiple sclerosis. OECs appear to migrate within the adult mammalian central nervous system (CNS) in animal models of spinal cord injury, but until recently there has been no systematic examination of the factors inducing or guiding this migration. Previous work in our lab (V.Skihar) implicated microglial reactivity in the generation of a migratory signal(s) inducing OECs to migrate towards an ethidium bromide-induced focal demyelination in the adult rat spinal cord. The long-term objective of this research project was to test the hypothesis that reactive microglial provide a migratory signal(s) driving the migration of OECs within the spinal cord of adult rats.<p>The first set of experiments determined the time-frame in which Wallerian degeneration (WD) induced microglial reactivity occurs in the right dorsal corticospinal tract (dCST) of adult rats at the level of T11 following aspiration of the contralateral sensorimotor cortex. This timing data from this study demonstrated a prominent microglial activation in the right dCST of T11 eight weeks after sensorimotor cortex injury indicating the microglial response to WD of dCST axons was very slow to appear. The second set of experiments determined whether OECs were induced to migrate in response to WD-induced microglial reactivity in the dCST, which based on the first set of experiments was known to occur within 8 weeks of lesioning the left sensorimotor cortex. This second set of experiments also examined the migratory path taken by OECs with respect to the location of reactive microglia (i.e. inside vs outside the right dCST). For these experiments, the left sensorimotor cortex was damaged 8 weeks prior to grafting the OECs at T12. <p>The next group of experiments examined the contribution of TNF-á induced microglial reactivity to generation of a migratory signal. First we identified concentrations of TNF-á that when injected into the DF of the T11 spinal cord segment of an adult rat induced microglial reactivity either along at least a 5 mm distance from the injection site or confined to the immediate vicinity of the injection site. The result of this experiment identified a concentration of 1 ng/µl and 0.01 ng/µl TNF-á as appropriate concentrations to induce the appropriate amount of microglial reactivity, respectively. The final set of experiments used these two concentrations to determine whether TNF-á induced microglial reactivity that is initiated 5 mm rostral to a DiI+ve OEC graft generates a migratory signal(s) inducing OECs to migrate towards the rostral part of T11 and whether the migratory signal(s) was present only if the microglial reactivity extended the full 5 mm distance between the TNF-á injection and the OEC graft. <p>The major findings were: i) there was a significantly higher density of DiI+ve OECs within the right dCST of rats in which there was WD-induced microglial reactivity as compared to the right dCST of rats in which there was no microglial reactivity; ii) the migratory path taken by DiI+ve OECs was preferentially within areas containing reactive microglia (i.e. dCST) and towards the site of TNF-á induced microglial reactivity (i.e. rostral to cell graft as opposed to caudal); iii) significantly more DiI+ve OECs migrated towards the site of a TNF-á injection when the microglia were reactive along the entire length of the migratory path between the cytokine injection and cell graft; and iv) minocycline treatment both dampened microglial reactivity and significantly reduced the number of migrating DiI+ve OECs. The major conclusions are that the migration of OECs within the adult rat spinal cord occurs in response to migratory signal(s) arising as a result of microglial activation and that this migration occurs preferentially along the path of microglial reactivity.

Olfactory ensheathing cells (OECs) are glial cells that are an attractive candidate for neural repair after spinal cord injury and for remyelination of axons in diseases such as multiple sclerosis. OECs appear to migrate within the adult mammalian central nervous system (CNS) in animal models of spinal cord injury, but until recently there has been no systematic examination of the factors inducing or guiding this migration. Previous work in our lab (V.Skihar) implicated microglial reactivity in the generation of a migratory signal(s) inducing OECs to migrate towards an ethidium bromide-induced focal demyelination in the adult rat spinal cord. The long-term objective of this research project was to test the hypothesis that reactive microglial provide a migratory signal(s) driving the migration of OECs within the spinal cord of adult rats.<p>The first set of experiments determined the time-frame in which Wallerian degeneration (WD) induced microglial reactivity occurs in the right dorsal corticospinal tract (dCST) of adult rats at the level of T11 following aspiration of the contralateral sensorimotor cortex. This timing data from this study demonstrated a prominent microglial activation in the right dCST of T11 eight weeks after sensorimotor cortex injury indicating the microglial response to WD of dCST axons was very slow to appear. The second set of experiments determined whether OECs were induced to migrate in response to WD-induced microglial reactivity in the dCST, which based on the first set of experiments was known to occur within 8 weeks of lesioning the left sensorimotor cortex. This second set of experiments also examined the migratory path taken by OECs with respect to the location of reactive microglia (i.e. inside vs outside the right dCST). For these experiments, the left sensorimotor cortex was damaged 8 weeks prior to grafting the OECs at T12. <p>The next group of experiments examined the contribution of TNF-á induced microglial reactivity to generation of a migratory signal. First we identified concentrations of TNF-á that when injected into the DF of the T11 spinal cord segment of an adult rat induced microglial reactivity either along at least a 5 mm distance from the injection site or confined to the immediate vicinity of the injection site. The result of this experiment identified a concentration of 1 ng/µl and 0.01 ng/µl TNF-á as appropriate concentrations to induce the appropriate amount of microglial reactivity, respectively. The final set of experiments used these two concentrations to determine whether TNF-á induced microglial reactivity that is initiated 5 mm rostral to a DiI+ve OEC graft generates a migratory signal(s) inducing OECs to migrate towards the rostral part of T11 and whether the migratory signal(s) was present only if the microglial reactivity extended the full 5 mm distance between the TNF-á injection and the OEC graft. <p>The major findings were: i) there was a significantly higher density of DiI+ve OECs within the right dCST of rats in which there was WD-induced microglial reactivity as compared to the right dCST of rats in which there was no microglial reactivity; ii) the migratory path taken by DiI+ve OECs was preferentially within areas containing reactive microglia (i.e. dCST) and towards the site of TNF-á induced microglial reactivity (i.e. rostral to cell graft as opposed to caudal); iii) significantly more DiI+ve OECs migrated towards the site of a TNF-á injection when the microglia were reactive along the entire length of the migratory path between the cytokine injection and cell graft; and iv) minocycline treatment both dampened microglial reactivity and significantly reduced the number of migrating DiI+ve OECs. The major conclusions are that the migration of OECs within the adult rat spinal cord occurs in response to migratory signal(s) arising as a result of microglial activation and that this migration occurs preferentially along the path of microglial reactivity.

Over the recent years, olfactory ensheathing cells (OECs) have gained world-wide attention due to their reputed potential in promoting spinal cord regeneration and repair. In order to isolate, identify, and characterize OECs in vitro and following implantation, researchers have used three OEC markers: p75NTR, GFAP, and S100. The downfall with using these specific proteins is that Schwann cells, which are located within the olfactory system, as well as migrate into the damaged spinal cord, also express these proteins. It is therefore impossible to distinguish OECs from phenotypically similar Schwann cells using these molecular markers. Recently proteomic analyses have revealed that OECs (derived from embryonic rat olfactory bulbs), but not Schwann cells (derived from adult rat sciatic nerves) express a variety of proteins. The main aim of this project is to determine if heat shock protein-27 (Hsp27), carbonic anhydrase-III (CA-III), and annexin-A3 (Anx3) markers label OECs but not Schwann cells, both in vivo and in vitro. Additional analyses were also done to determine if smooth muscle α-actin (SMA) and calponin (two smooth muscle-related markers previously shown to label mucosal OECs of adult rats) label bulbar OECs of adult rats and OECs of adult cats. Using immunohistochemistry we found that SMA labeled olfactory mucosal and bulbar OECs of adult rats and adult cats, Hsp27 labeled olfactory mucosal and bulbar OECs of adult rats and olfactory mucosal OECs of adult cats, while calponin labeled only olfactory mucosal OECs of adult rats. In addition, calponin and SMA did not label Schwann cells (in vivo and in vitro), while Hsp27 labeled this peripheral glial cell. Finally, CA-III did not label OECs of adult rats or adult cats, in vivo or in vitro, and Anx3 did not label OECs in vivo, but showed immunopositive labeling of OECs and Schwann cells in vitro. In conclusion, Hsp27, CA-III, and Anx3 cannot be used as OECs markers either because of their expression in both OECs and Schwann cells or their lack of expression in OECs. Discovering new molecular markers expressed only by OECs is essential in order to determine the properties, fate, and overall potential of OECs in promoting spinal cord regeneration. / Thesis (Master, Neuroscience Studies) -- Queen's University, 2008-09-29 09:50:09.869

Olfactory ensheathing cells (OECs), the glia of the olfactory nerve, are promising candidates for patient-specific cell-mediated repair of both peripheral nerves and the spinal cord. The recent discovery that OECs originate from the neural crest, rather than the olfactory epithelium as previously thought, potentially means that homogeneous populations of OECs for repair could be expanded in culture from neural crest stem cells persisting in the patient's own skin and hair follicles. The first step towards this long-term goal is to understand the molecular mechanisms underlying neural crest differentiation into OECs, as opposed to Schwann cells (the glia of all other peripheral nerves), which are less effective in spinal cord repair. To identify transcription factors and signalling pathways that might be involved in OEC versus Schwann cell differentiation, I took an unbiased transcriptome profiling approach. Taking advantage of Sox10 expression throughout both OEC and Schwann cell development, I used laser-capture microdissection on cryosections of mouse embryos carrying a Sox10:H2BVenus transgene, to isolate OEC subpopulations (olfactory mucosal OECs, from the olfactory nerve, and olfactory nerve layer OECs, from the olfactory nerve layer surrounding the olfactory bulb) at different stages of development, and Schwann cells from trigeminal nerve branches on the same sections, for RNA-seq and cross-wise comparison of transcriptomes. Validation of candidate genes by in situ hybridisation revealed some contamination with adjacent cells from mesenchyme, olfactory epithelium or olfactory bulb, but also identified the expression in developing OECs of various genes previously reported to be expressed in adult OECs, and of over 20 genes previously unknown in OECs. Some of these genes are expressed by OECs but not Schwann cells; some are expressed by olfactory nerve layer OECs but not olfactory mucosal OECs, while some are expressed by olfactory mucosal OECs and Schwann cells but not olfactory nerve layer OECs. For a subset of the genes, I was also able to analyse OEC differentiation in mouse mutants. I also collected transcriptome data from neural crest-derived cells that persist on the olfactory nerve in Sox10-null embryos (in which neural crest-derived cells colonise the olfactory nerve, but normal OEC differentiation is disrupted). Comparison with wild-type OEC transcriptome data from the same embryonic stage identified genes whose expression is likely either downregulated or up-regulated in the absence of Sox10, supporting a role in normal OEC differentiation. Overall, these various transcriptomic comparisons (between OECs at different developmental stages, different OEC subpopulations, OECs versus Schwann cells, and OECs versus Sox10-null neural crest-derived cells on the olfactory nerve) have identified multiple transcription factor and signalling pathway genes, amongst others, that are expressed during OEC development in vivo (including some specific to different OEC subpopulations) and that may be important for OEC differentiation. Furthermore, some of these genes are not expressed by embryonic Schwann cells. This work provides a foundation for understanding how to promote OEC rather than Schwann cell differentiation from neural crest stem cells in culture, with the potential for clinical application in the future.

The olfactory system exhibits remarkable regenerative ability in it’s neuronal population. The success of continuous neurogenesis is thought to be due, at least in part, to its unique glia – olfactory ensheathing cells (OECs). OECs bear characteristics of both peripheral and central glia, and serve to ensheath, guide and promote growth of olfactory receptor neurons (ORNs) throughout both development and adult life. Brain lipid binding protein (BLBP) is most highly expressed by radial glia during embryonic development. It is largely down-regulated in the adult CNS, but BLBP expression is retained in the adult by special subpopulations of glia, including OECs. BLBP expression is induced in radial glia via Notch signaling, but it is not known if these same mechanisms regulate BLBP expression in the adult CNS. Axonal-glial signaling is a dynamic process whereby closely apposed neuronal and glial cells regulate the growth, maintenance and plasticity of one another through direct cell-cell signaling. Delta/Notch-like EGF-related receptor (DNER) is a transmembrane protein expressed by Purkinje cells which has been implicated in the regulation of BLBP in Bergmann glia during cerebellum development through Notch1 deltex-dependent non-canonical signaling. We have found that DNER is expressed in more mature ORNs, and other exclusive subpopulations of cells within the CNS. OECs in close apposition with DNER-expressing ORNs in vivo appear to maintain the highest BLBP expression found in the nervous system through development and adulthood. Immunofluorescence shows that this close relationship between BLBP expressing cells and DNER expressing cells also appears to be retained in specialized areas such as the hippocampus, retina and spinal cord, throughout mouse CNS development as well as in the mature system. Removing DNER or axonal input in vivo decreases the robustness of OEC BLBP expression, and the number of cells in OEC culture expressing BLBP decreases rapidly with time. OEC co-culture with a DNER expressing monolayer increases the number of OECs in vitro which express BLBP, providing evidence for the regulation of BLBP expression in OECs by DNER expression in apposing ORNs.

The olfactory system exhibits remarkable regenerative ability in it’s neuronal population. The success of continuous neurogenesis is thought to be due, at least in part, to its unique glia – olfactory ensheathing cells (OECs). OECs bear characteristics of both peripheral and central glia, and serve to ensheath, guide and promote growth of olfactory receptor neurons (ORNs) throughout both development and adult life. Brain lipid binding protein (BLBP) is most highly expressed by radial glia during embryonic development. It is largely down-regulated in the adult CNS, but BLBP expression is retained in the adult by special subpopulations of glia, including OECs. BLBP expression is induced in radial glia via Notch signaling, but it is not known if these same mechanisms regulate BLBP expression in the adult CNS. Axonal-glial signaling is a dynamic process whereby closely apposed neuronal and glial cells regulate the growth, maintenance and plasticity of one another through direct cell-cell signaling. Delta/Notch-like EGF-related receptor (DNER) is a transmembrane protein expressed by Purkinje cells which has been implicated in the regulation of BLBP in Bergmann glia during cerebellum development through Notch1 deltex-dependent non-canonical signaling. We have found that DNER is expressed in more mature ORNs, and other exclusive subpopulations of cells within the CNS. OECs in close apposition with DNER-expressing ORNs in vivo appear to maintain the highest BLBP expression found in the nervous system through development and adulthood. Immunofluorescence shows that this close relationship between BLBP expressing cells and DNER expressing cells also appears to be retained in specialized areas such as the hippocampus, retina and spinal cord, throughout mouse CNS development as well as in the mature system. Removing DNER or axonal input in vivo decreases the robustness of OEC BLBP expression, and the number of cells in OEC culture expressing BLBP decreases rapidly with time. OEC co-culture with a DNER expressing monolayer increases the number of OECs in vitro which express BLBP, providing evidence for the regulation of BLBP expression in OECs by DNER expression in apposing ORNs.

The olfactory system exhibits remarkable regenerative ability in it’s neuronal population. The success of continuous neurogenesis is thought to be due, at least in part, to its unique glia – olfactory ensheathing cells (OECs). OECs bear characteristics of both peripheral and central glia, and serve to ensheath, guide and promote growth of olfactory receptor neurons (ORNs) throughout both development and adult life. Brain lipid binding protein (BLBP) is most highly expressed by radial glia during embryonic development. It is largely down-regulated in the adult CNS, but BLBP expression is retained in the adult by special subpopulations of glia, including OECs. BLBP expression is induced in radial glia via Notch signaling, but it is not known if these same mechanisms regulate BLBP expression in the adult CNS. Axonal-glial signaling is a dynamic process whereby closely apposed neuronal and glial cells regulate the growth, maintenance and plasticity of one another through direct cell-cell signaling. Delta/Notch-like EGF-related receptor (DNER) is a transmembrane protein expressed by Purkinje cells which has been implicated in the regulation of BLBP in Bergmann glia during cerebellum development through Notch1 deltex-dependent non-canonical signaling. We have found that DNER is expressed in more mature ORNs, and other exclusive subpopulations of cells within the CNS. OECs in close apposition with DNER-expressing ORNs in vivo appear to maintain the highest BLBP expression found in the nervous system through development and adulthood. Immunofluorescence shows that this close relationship between BLBP expressing cells and DNER expressing cells also appears to be retained in specialized areas such as the hippocampus, retina and spinal cord, throughout mouse CNS development as well as in the mature system. Removing DNER or axonal input in vivo decreases the robustness of OEC BLBP expression, and the number of cells in OEC culture expressing BLBP decreases rapidly with time. OEC co-culture with a DNER expressing monolayer increases the number of OECs in vitro which express BLBP, providing evidence for the regulation of BLBP expression in OECs by DNER expression in apposing ORNs. / Medicine, Faculty of / Graduate

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