Mi Chuah

Olfactory ensheathing cells promote collateral axonal branching in the injured adult rat spinal cord

In recent years, injection of olfactory ensheathing cells (ECs) into the spinal cord has been used as an experimental strategy to promote regeneration of injured axons. In this study, we have compared the effects of transplanting encapsulated ECs with those injected directly into the spinal cord. The dorsal columns of adult rats were cut at T(8-9) and rats in experimental groups received either EC-filled porous polymer capsules or culture medium (CM)-filled capsules with ECs injected at the injury site. Control rats were in three groups: (1) uninjured, (2) lesion with transplantation of CM-filled capsules and (3) lesion with transplantation of CM-filled capsules and injections of CM. Three weeks after injury, Fluororuby was injected into the hindlimb motor and somatosensory cortex to label corticospinal neurons. Observations indicated that there were a few regenerating fibres, up to 10, in the EC-treated groups. In rats that received encapsulated ECs, regenerating fibres were present in close association with the capsule. Rats that received EC injections demonstrated a significant increase in the number of collateral branches from the intact ventral corticospinal tract (vCST) compared with the corresponding control, CM-injected group (P=0.003), while a trend for increased collateral branches was observed in rats that received encapsulated ECs (P=0.07).

Olfactory ensheathing cells promote neurite sprouting of injured axons in vitro by direct cellular contact and secretion of soluble factors

Olfactory ensheathing cells (OECs) represent an exciting possibility for promoting axonal regeneration within the injured spinal cord. A number of studies have indicated the ability of these cells to promote significant reactive sprouting of injured axons within the injured spinal cord, and in some cases restoration of functional abilities. However, the cellular and/or molecular mechanisms OECs use to achieve this are unclear. To investigate such mechanisms, we report for the first time the ability of OECs to promote post-injury neurite sprouting in an in vitro model of axonal injury. Using this model, we were able to differentiate between the direct and indirect mechanisms underlying the ability of OECs to promote neuronal recovery from injury. We noted that OECs appeared to act as a physical substrate for the growth of post-injury neurite sprouts. We also found that while post-injury sprouting was promoted most when OECs were allowed to directly contact injured neurons, physical separation using tissue culture inserts (1 mm pore size, permeable to diffusible factors but not cells) did not completely block the promoting properties of OECs, suggesting that they also secrete soluble factors which aid post-injury neurite sprouting. Furthermore, this in vitro model allowed direct observation of the cellular interactions between OECs and sprouting neurites using live-cell-imaging techniques. In summary, we found that OECs separately promote neurite sprouting by providing a physical substrate for growth and through the expression of soluble factors. Our findings provide new insight into the ability of OECs to promote axonal regeneration, and also indicate potential targets for manipulation of these cells to enhance their restorative ability.

Basic fibroblast growth factor in the primary olfactory pathway: mitogenic effect on ensheathing cells.

The mitogenic effect of basic fibroblast growth factor and nerve growth factor (2.5S) on olfactory ensheathing cell culture was examined by bromodeoxyuridine uptake. It was found that, at 10 ng/ml, basic fibroblast growth factor elicited about a three-fold increase in proliferation, while the stimulatory effect of nerve growth factor was considerably less. The increased proliferation resulting from basic fibroblast growth factor could be attributed to perlecan, which was shown to be expressed by ensheathing cell in culture. Perlecan is known to induce high-affinity binding of basic fibroblast growth factor to receptors on cell membranes. Immunohistochemical staining demonstrated that basic fibroblast growth factor was abundantly expressed in select regions of the lamina propria underlying the olfactory epithelium. In these regions, contiguous patches of olfactory epithelium also showed the presence of basic fibroblast growth factor. Although basic fibroblast growth factor was present on the periphery of nerve bundles in the olfactory nerve layer of the bulb, all other laminae did not demonstrate the presence of this factor. The immunohistochemistry and cell culture results show that regions of the lamina propria and small patches of the olfactory epithelium, by their presence of basic fibroblast growth factor, are potential sites of ensheathing cell proliferation in vivo.

Ultrastructural study of ensheathing cells in early development of olfactory axons.

Ultrastructural observations in the grey short-tailed opossum (Monodelphis domestica) and rat revealed that ensheathing cells were intimately related to the early formation of olfactory axons. Whilst the axons were still in the olfactory epithelium, they were enveloped by ensheathing cell processes which formed a cradle-like structure on the basal side of the epithelium. Continued downgrowth of the axons towards the lamina propria resulted in an evagination with the ensheathing cell process or cell body in direct contact with the basal lamina. Subsequently the basal lamina became fragmented, and the newly formed olfactory nerve emerged from the olfactory epithelium. As the olfactory nerve grew, it was observed that the ensheathing cell process always extended ahead of the axons while axon terminals moving ahead of ensheathing cells were never observed. The findings in this study suggest that ensheathing cells play a role in regulating and promoting olfactory axon growth.

Metallothionein-III Inhibits Initial Neurite Formation in Developing Neurons as Well as Postinjury, Regenerative Neurite Sprouting

Human metallothionein-III (MT-III) is an inhibitory factor deficient in the Alzheimers disease (AD) brain. MT-III has been identified as an inhibitor of neurite sprouting, and its deficiency has been proposed to be involved in the formation of neurofibrillary tangles (NFT) in the neuropathology of AD. However, there has been limited investigation of the proposed neurite growth inhibitory properties of MT-III. We have applied recombinant human MT-III to both single cell embryonic cortical neurons (to investigate initial neurite formation), as well as mature (21 days postplating) clusters of cortical neurons (to investigate the regenerative sprouting response following injury). We report that MT-III inhibited the initial formation of neurites by rat embryonic (E18) cortical neurons. This was based on both the percentage of neurite positive neurons and the number of neurites per neuron (45 and 30% inhibition, respectively). Neurite inhibition was only observed in the presence of adult rat brain extract, and was also reversible following replacement of MT-III-containing medium. MT-III inhibited the formation and growth of both axons and dendrites. Of more physiological significance, MT-III also inhibited the regenerative neurite sprouting response following axonal transection. The morphology of sprouting neurites was also altered, with the distal tip often ending in bulb-like structures. Based on these results, we propose that MT-III, in the presence of brain extract, is a potent inhibitor of neurite sprouting, and may be involved in abnormal sprouting potentially underlying both AD and epilepsy.

Olfactory ensheathing cells are attracted to, and can endocytose, bacteria

Abstract.Olfactory ensheathing cells (OECs) have been shown previously to express Toll-like receptors and to respond to bacteria by translocating nuclear factor-κB from the cytoplasm to the nucleus. In this study, we show that OECs extended significantly more pseudopodia when they were exposed to Escherichia coli than in the absence of bacteria (p=0.019). Co-immunoprecipitation showed that E. coli binding to OECs was mediated by Toll-like receptor 4. Lyso-Tracker, a fluorescent probe that accumulates selectively in lysosomes, and staining for type 1 lysosome-associated membrane proteins demonstrated that endocytosed FITC-conjugated E. coli were translocated to lysosomes. They appeared to be subsequently broken down, as shown by transmission electron microscopy. No obvious adherence to the membrane and less phagocytosis was observed when OECs were incubated with inert fluorescent microspheres. The ability of OECs to endocytose bacteria supports the notion that OECs play an innate immune function by protecting olfactory tissues from bacterial infection.

Effects of astrocyte implantation into the hemisected adult rat spinal cord

Morphological and biochemical methods were applied to assess the effects of implanting cultured astrocytes into the hemisected adult rat spinal cord. Astrocytes were purified from neonatal rat cortex and introduced into the lesioned spinal cord either in suspension injection or cultured on gelfoam first. The control groups were rats which had hemisection with injection of culture media or with gelfoam grafted alone. At various time points after surgery (two weeks to two months), the spinal cord was removed and processed for routine light microscopy, immunofluorescence, gel electrophoresis and immunoblotting. As early as two weeks after surgery, a significantly smaller volume of scar tissue was consistently found in the experimental groups. This reduced scarring was also confirmed by immunofluorescence staining and immunoblotting for glial fibrillary acidic protein in the specimens two months after hemisection. Compared to the control groups, the experimental groups also had more intense staining for neurofilaments, which was confirmed by immunoblotting. However, labelling of the astrocytes with Phaseolus vulgaris leucoagglutinin conjugated with fluorescein showed that the astrocytes migrated at a rate of 0.6 mm/day from the original implanted site. The results therefore suggested that the cultured astrocytes probably exerted their effects over a short time period (less than two weeks) around the lesion site. They could have altered the microenvironment and as a result less scar tissue was formed. Hence, there was less barrier to the regrowth of nerve fibres.

Glial growth factor 2 induces proliferation and structural changes in ensheathing cells

Ensheathing cells were isolated from neonatal rat olfactory bulbs and cultured in the presence of glial growth factor 2 (GGF2). Proliferation assay showed that at concentrations of up to 60 ng/ml GGF2, ensheathing cells underwent a modest increase in proliferation rate. This stimulation was not maintained at high doses of GGF2 at 100 ng/ml or more. Chemotaxis chambers and scanning electron microscopy were used to determine whether GGF2 was a chemoattractant for ensheathing cells. Although the results showed no chemotactic response to GGF2, ensheathing cells demonstrated structural changes when cultured in the presence of 20 ng/ml GGF2. Ultrastructural observations revealed that GGF2 promoted increased deposition of extracellular matrix on the cell membrane, more cytoskeletal elements in the processes and as a possible consequence, contributed to a more rigid support. Ensheathing cells cultured in the absence of GGF2 often extended thinner and curved processes. Reverse transcription-polymerase chain reaction confirmed the presence of GGF2 transcripts in ensheathing cells, suggesting that ensheathing cells themselves are a source of GGF2.

Olfactory ensheathing cell phenotype following implantation in the lesioned spinal cord

Although olfactory ensheathing cells (OECs) are used to promote repair in the injured spinal cord, little is known of their phenotype in this environment. In this study, using quantitative reverse transcriptase-polymerase chain reaction RT-PCR, expression of neuregulin-1 mitogen/survival factors and the axonal growth regulator Nogo was quantified in OECs and compared with other non-neuronal cells. Their expression was also compared with OECs which had previously been encapsulated in a porous polymer tube and implanted into the injured spinal cord. Similar to astrocytes and fibroblasts, OECs expressed various neuregulin subtypes including neu differentiation factor, glial growth factor and sensory and motorneuron-derived factor. Implanted OECs upregulated neu differentiation factor and secreted neuregulin, but downregulated expression of all other variants. OECs and oligodendrocytes expressed Nogo-A, -B and -ABC and were immunopositive for Nogo-A protein. The Nogo-A protein in OECs was found to be cytoplasmic rather than nuclear or cell surface associated. Unlike oligodendrocytes, OECs expressed Nogo-66 receptor (NgR) mRNA. Implanted OECs upregulated Nogo-A and -B, but downregulated Nogo-ABC and NgR.

Excitotoxicity mediated by non-NMDA receptors causes distal axonopathy in long-term cultured spinal motor neurons

Excitotoxicity has been implicated as a potential cause of neuronal degeneration in amyotrophic lateral sclerosis (ALS). It has not been clear how excitotoxic injury leads to the hallmark pathological changes of ALS, such as the abnormal accumulation of filamentous proteins in axons. We have investigated the effects of overactivation of excitatory receptors in rodent neurons maintained in long‐term culture. Excitotoxicity, mediated principally via non‐N‐methyl‐d‐aspartate (NMDA) receptors, caused axonal swelling and accumulation of cytoskeletal proteins in the distal segments of the axons of cultured spinal, but not cortical, neurons. Axonopathy only occurred in spinal neurons maintained for 3 weeks in vitro, indicating that susceptibility to axonal pathology may be related to relative maturity of the neuron. Excitotoxic axonopathy was associated with the aberrant colocalization of phosphorylated and dephosphorylated neurofilament proteins, indicating that disruption to the regulation of phosphorylation of neurofilaments may lead to their abnormal accumulation. These data provide a strong link between excitotoxicity and the selective pattern of axonopathy of lower motor neurons that underlies neuronal dysfunction in ALS.