Loss of directed fore-limb reaching after destruction of spinal grey matter.

The use of the ipsilateral fore-paw to retrieve food pellets was studied in 55 rats with unilateral lesions in the dorsal spinal cord at the C1/2 level. Of these rats 30 either retained (16) or recovered (14) function within 25 days after the lesion. The remaining 25 rats showed no return of function over an 8 week postoperative testing period. Plotting the extent of the lesions showed that all 30 rats showing retrieval had complete destruction of the main dorsal corticospinal tract in the dorsal columns and some damage to the adjacent medial part of the grey matter of Rexed's laminae V to VII, but sparing the dorsolateral CST and more than half of the grey matter. In the 25 rats with no return of function the lesions also included extensive destruction through the medio-lateral extent of the grey matter.

Transplanted olfactory ensheathing cells incorporated into the optic nerve head ensheathe retinal ganglion cell axons: possible relevance to glaucoma.

A mixture of olfactory ensheathing cells and fibroblasts cultured from the adult rat olfactory mucosa was transplanted through a scleral incision into the retina. A major stream of transplanted cells migrated through the stratum opticum and penetrated for up to about 0.5mm into the optic nerve head. This stream of transplanted cells consisted of a mixture of bipolar olfactory ensheathing cells with long processes which give rise to a non-myelinating ensheathment of single retinal ganglion cell axons, and olfactory nerve fibroblasts embedded in a dense fibronectin-positive extracellular matrix. A second stream of ovoid olfactory ensheathing cells with tufted processes and unaccompanied by fibroblasts or matrix migrate into the internal plexiform layer. The incorporation of olfactory ensheathing cells in the optic nerve head may suggest future possibilities for protection of the axons in this vulnerable region from mechanical damage, as in the raised intraocular pressure of glaucoma.

Olfactory ensheathing cells in the nasal mucosa of the rat and human.

Olfactory ensheathing cells are unique glial cells which have the ability to encourage nerve fibres to grow, and may have potential for use as a therapy for spinal cord and brachial plexus injuries. These images show olfactory ensheathing cells in the olfactory mucosa of the nose, and provide insight into their function. The olfactory mucosa is likely to be a convenient source for these cells in future clinical studies of CNS repair.

International spinal research trust research strategy. III: A discussion document.

STUDY DESIGN: Discussion document. OBJECTIVES/METHODS: To review the Research Strategy of the International Spinal Research Trust (ISRT), which identifies key areas of basic and clinical research that are likely to be beneficial in developing potential treatments for spinal cord injury for funding. This strategy is intended to both guide the programme of research towards areas of priority and stimulate discussion of the different avenues of research. This latest document has been developed to take into account the scientific progress in the 6 years since publication of the previous Research Strategy. RESULTS/DISCUSSION: The latest scientific developments in research designed to repair the spinal cord and restore function following injury and how they might impact on spinal cord injury research are highlighted.

Repair of spinal cord injury: ripples of an incoming tide, or how I spent my first 40 years in research.

Abstract of the inaugural lecture on appointment to the Chair of Neural Regeneration at University College London January 2006. Record of personal research. Electron microscopic observations led to the concept that the adult brain is capable of forming new synapses after injury, and the search for methods to repair brain and spinal cord injuries. It is proposed that the failure of regeneration after central axotomy is due to protective glial scarring leading to the loss of the aligned astrocytic pathways needed for axon elongation. Taking advantage of the discovery that the adult olfactory system is capable of continual renewal, cultured olfactory ensheathing cells were transplanted into lesions of the spinal cord and spinal roots. The transplants re-opened scarred glial pathways, allowed the regeneration of severed nerve fibres, and the restoration of various functions, including paw reaching, climbing, and supraspinal respiratory impulses to the phrenic nerve.

After facial nerve damage, regenerating axons become aberrant throughout the length of the nerve and not only at the site of the lesion: an experimental study.

After facial nerve trauma, aberrant regeneration is associated with synkinesis. Animal models of mechanical nerve guides or reparative cell transplants at the site of a lesion have not been shown to improve disorganized regeneration. We examined whether this is because regenerating axons become disorganized throughout the length of the nerve and not only at the site of the lesion. In rats (n = 12), retrograde fluorescent tracer techniques were used to establish that most of the temporal branch fibres were carried in the superior half of the facial nerve trunk. In two further groups of rats (n = 24) a complete proximal facial nerve lesion was made, and the nerve immediately repaired by suture. After 4 weeks, at a second operation, the superior half of the facial nerve trunk was cut, either proximal or distal to the original lesion, and retrograde tracers were applied to distal branches of the nerve. It was possible to localize the points at which regenerating fibres became aberrant in their course by studying the number of labelled motoneurons in the facial nucleus after application of the tracer to the temporal branch of the nerve: this was similar in the distal and proximal hemisection groups, suggesting that aberrant axonal development occurred throughout the length of the nerve. Future strategies aimed at improving the organization of regeneration need to provide guidance cues not only at the site of the lesion as previously thought, but also throughout the length of the nerve.

Immune rejection of a facial nerve xenograft does not prevent regeneration and the return of function: an experimental study.

Nerve grafts may be used to repair damaged peripheral nerves and also to facilitate spinal cord regeneration after experimental trauma. Little is known, however, about the possible use of xenografts and the role of immune rejection in the outcome of repair. In rats, excision of a short (7-8 mm) segment of facial nerve at its exit point from the skull base results in a permanent deficit in eye closure in the blink reflex. This deficit can be repaired by transplantation of a segment of either syngeneic rat facial nerve or xenogeneic Balb-C mouse sciatic nerve either with or without cyclosporine immunosuppression. With longer (15-20 mm) transplants, however, restoration of eye closure becomes dependent on cyclosporine administration. Thus, in a situation where nerve repair does not occur without a graft, a host immune attack has an attritional effect which is not sufficient to prevent repair over short distances, but becomes obvious when the regenerating fibres have to cross longer segments of transplanted tissue.

Ensheathment of the olfactory nerves in the adult rat.

The ensheathment of the olfactory nerve fibres is achieved by cooperation of two cell types. The olfactory ensheathing cells have a rounded outer surface enclosed in a continuous single basal lamina, and enclose an inner compartment from which overlapping processes of the same and adjacent cells enwrap interweaving territories of tightly apposed aligned axons. The olfactory nerve fibroblasts are highly flattened, dense cells generating multiple layers of very thin processes encircling individual or groups of olfactory ensheathing cells. This paper illustrates the unique ultrastructural features of this ensheathment.

Fluorescent retrograde neuronal tracers that label the rat facial nucleus: a comparison of Fast Blue, Fluoro-ruby, Fluoro-emerald, Fluoro-Gold and DiI.

Many fluorescent retrograde tracers are commercially available for neuroanatomical studies. They have been used with varying success in different models and can be very effective in the study of the facial nerve and nucleus. We compare the tracers Fast Blue (FB), Fluoro-ruby, Fluoro-emerald, Fluoro-Gold (FG), and DiI in the rat facial nucleus after application to the buccal division of the nerve. There were no significant differences between counts of cells on the left and right sides of the brain stem with any of the tracers. FB produced a lower sample variation than the other tracers, and together with DiI, demonstrated greater axonal labelling when applied to the surface of the epineurium. FB and FG resulted in strong retrograde labelling of the facial nucleus after only 2 days from injection. All the tracers produced adequate cell labelling after 1 week from nerve application. This labelling persisted for up to 8 weeks for most tracers except FG, which did not produce satisfactory labelling at 8 weeks.

How do transplanted olfactory ensheathing cells restore function?

In this article, we review our work on regeneration of the corticospinal tract in rats following a lesion at upper cervical level. We outline the rationale for using olfactory ensheathing cells, and summarize the evidence for regeneration and functional recovery. The present interpretation on the mechanisms of functional recovery is partly hypothetical, and we emphasize where further experimental evidence is needed.

Olfactory ensheathing cells - another miracle cure for spinal cord injury?

Several recent publications describe remarkably promising effects of transplanting olfactory ensheathing cells as a potential future method to repair human spinal cord injuries. But why were cells from the nose transplanted into the spinal cord? What are olfactory ensheathing cells, and how might they produce these beneficial effects? And more generally, what do we mean by spinal cord injury? To what extent can we compare repair in an animal to repair in a human?

Columns of Schwann cells extruded into the CNS induce in-growth of astrocytes to form organized new glial pathways.

Our previous work showed that stereotaxic microextrusion of columns of purified peripheral nerve-derived Schwann cells into the thalamus of syngeneic adult rats induces host axons to grow into the column and form a new fiber tract. Here we describe the time course of cellular events that lead to the formation of this new tract. At 2 h postoperation, numerous OX42-positive microglia accumulated at the graft-host interface, after which donor columns became progressively and heavily infiltrated by microglia/macrophages that took on an elongated morphology in parallel with the highly orientated processes of the donor Schwann cells. The penetration of host astrocytic processes into the Schwann cell columns was substantially slower in onset, being first detected at 4 days postoperation. This event was contemporaneous with the in-growth of host thalamic axons. Between 7 and 14 days postoperation, GFAP-positive astrocytes became fully incorporated into the transplants, where they too adopted an elongated form, orientated in parallel with the longitudinal axis of the graft. Thus, the columns became a mosaic of elongated and highly orientated donor Schwann cells intimately mingled with host microglia, astrocytes, and numerous, largely unbranched 200-kDa neurofilament-positive axons from the adjacent thalamus. Electron microscopy demonstrated that the processes of donor Schwann cells and host astrocytes within the column formed tightly packed bundles that were surrounded by a partial or complete basal lamina. Control columns, formed by extruding freeze-thaw-killed Schwann cells or purified peripheral nerve fibroblasts induced a reactive injury response by the adjacent host microglia and astrocytes, but neither host astrocytes nor neurofilament-positive axons were incorporated into the columns. A better understanding of the mechanisms that regulate the interactions between donor and host glia should facilitate improved integration of such grafts and enhance their potential for inducing tissue repair.

Repair of corticospinal axons by transplantation of olfactory ensheathing cells.

This paper examines the possibility of repairing cut central connections by transplantation of glial cells which modify the glial pathways and take advantage of the inherent growth capacity in adult neurons. We found that transplants of cultured olfactory ensheathing cells into lesions of the adult rat corticospinal tract induced long growth of cut axons across the lesion. Acquisition of a directed forepaw reaching function was restored on the operated side.

Improved post-embedding immunocytochemistry of myelinated nervous tissue for electron microscopy.

The particularly high lipid content of normal mature adult myelin sheaths, together with the light fixation protocols usually necessary to retain antigenicity, combine to make white matter nervous tissue an especially problematical subject for post-embedding immuno-electron microscopy using modern acrylic resins. Fixation and infiltration modifications to standard processing schedules for Lowicryl were found to greatly improve the embedding and therefore the resulting morphology. This in turn improved the signal to noise ratio by reducing the high non-specific backgrounds usually found in poorly infiltrated areas. Using Lowicryl HM20, we have been able to obtain satisfactory immunostaining for myelin basic protein with good retention of structural integrity in the myelin of both normal and lesioned adult cortico spinal tract.

Signalling by the RET receptor tyrosine kinase and its role in the development of the mammalian enteric nervous system.

RET is a member of the receptor tyrosine kinase (RTK) superfamily, which can transduce signalling by glial cell line-derived neurotrophic factor (GDNF) and neurturin (NTN) in cultured cells. In order to determine whether in addition to being sufficient, RET is also necessary for signalling by these growth factors, we studied the response to GDNF and NTN of primary neuronal cultures (peripheral sensory and central dopaminergic neurons) derived from wild-type and RET-deficient mice. Our experiments show that absence of a functional RET receptor abrogates the biological responses of neuronal cells to both GDNF and NTN. Despite the established role of the RET signal transduction pathway in the development of the mammalian enteric nervous system (ENS), very little is known regarding its cellular mechanism(s) of action. Here, we have studied the effects of GDNF and NTN on cultures of neural crest (NC)-derived cells isolated from the gut of rat embryos. Our findings suggest that GDNF and NTN promote the survival of enteric neurons as well as the survival, proliferation and differentiation of multipotential ENS progenitors present in the gut of E12.5-13.5 rat embryos. However, the effects of these growth factors are stage-specific, since similar ENS cultures established from later stage embryos (E14. 5-15.5), show markedly diminished response to GDNF and NTN. To examine whether the in vitro effects of RET activation reflect the in vivo function(s) of this receptor, the extent of programmed cell death was examined in the gut of wild-type and RET-deficient mouse embryos by TUNEL histochemistry. Our experiments show that a subpopulation of enteric NC undergoes apoptotic cell death specifically in the foregut of embryos lacking the RET receptor. We suggest that normal function of the RET RTK is required in vivo during early stages of ENS histogenesis for the survival of undifferentiated enteric NC and their derivatives.

Death of oligodendrocytes and microglial phagocytosis of myelin precede immigration of Schwann cells into the spinal cord.

Small, circumscribed electrolytic lesions were made in the upper cervical corticospinal tract in adult rats. In the centre of the lesion, the axons and all other tissue elements were totally destroyed. Surrounding this region of destruction is an area of tissue which is only partially damaged. In this area TUNEL positive staining of contiguous rows of tract glial cells indicates massive oligodendrocytic apoptosis at 1-3 days after operation, but axons, astrocytes and blood vessels survive. From around 4 days, the corticospinal axons in this area are demyelinated, and the microglia contain ingested myelin, identified in electron micrographs as characteristic MBP immunoreactive laminar cytoplasmic bodies. After around 3 weeks, large numbers of Schwann cells, continuous with those on the pial surface of the spinal cord, accumulate along the lesion track and selectively infiltrate the perilesional reactive area, where they mingle intimately with the phagocytic microglia. Electron micrographs show that at this time basal lamina-enclosed Schwann cell processes establish non-myelinated ensheathment of axons. From around 4 weeks after operation, prominent Schwann cell myelination is indicated by P0 immunoreactivity, and peripheral type, one-to-one myelination in electron micrographs. Thus the effect of the selective loss of oligodendrocytes is to first activate microglia, and then to induce a replacement of myelin by Schwann cells.

Regeneration of adult rat corticospinal axons induced by transplanted olfactory ensheathing cells.

Precisely localized focal stereotaxic electrolytic lesions were made in the corticospinal tract at the level of the first to second cervical segments in the adult rat. This consistently destroyed all central nervous tissue elements (axons, astrocytes, oligodendrocytes, microglia, and microvessels) in a highly circumscribed area. In a group of these rats immediately after lesioning, a suspension of cultured adult olfactory ensheathing cells was transplanted into the lesion site. Within the first week after transplantation, the cut corticospinal axons (identified by anterograde transport of biotin dextran) extended caudally along the axis of the corticospinal tract as single, fine, minimally branched sprouts that ended in a simple tip, often preceded by a small varicosity. By 3 weeks, the regenerating axons, ensheathed by P0-positive peripheral myelin had accumulated into parallel bundles, which now extended across the full length of the lesioned area and reentered the caudal part of the host corticospinal tract. The transplants contained two main types of cells: (1) p75-expressing S cells, which later formed typical peripheral one-to-one myelin sheaths around individual ensheathed axons, and (2) fibronectin-expressing A cells, which aggregated into tubular sheaths enclosing bundles of myelinated axons. The point of reentry of the axons into the central nervous territory of the caudal host corticospinal tract was marked by the resumption of oligodendrocytic myelination. Thus the effect of the transplant was to form a "patch" of peripheral-type tissue across which the cut central axons regenerated and then continued to grow along their original central pathway.

Embryonic entorhinal transplants partially ameliorate the deficits in spatial memory in adult rats with entorhinal cortex lesions.

Our previous studies have demonstrated that axons from grafts of embryonic entorhinal cortex (EC) can reinnervate the deafferented zones in the hippocampus and form synaptic connections with the host dentate gyrus in adult mice and rats deprived of their own entorhinal inputs. Here, we have examined the ability of the EC grafts to ameliorate deficits in spatial memory. Three months after transplantation, the grafted rats and control animals were subjected to Morris water maze testing followed by histological examination. According to the exact position of grafts in the host brain, the rats with lesion and EC transplants were divided into two groups, one with EC grafts contacting both the hippocampus and overlying neocortex (n=7, EC1) and another with EC grafts confined within the hippocampus (n=6, EC2). While EC2 rats were still as impaired as those with lesion and transplants of non-entorhinal cortex (n=10, NEC) or with lesions only (n=7, LES), the EC1 rats performed better than the LES group. In a spatial memory trial, the EC1 group made more crossings over platform site and showed more focused search behavior than EC2, LES, NEC groups. The data suggest that EC grafts could partially ameliorate the deficit in spatial learning behavior in the EC-lesioned adult rats. The requirement for the graft to contact both the neocortex and the hippocampus suggests that the functional effects may be exerted by the formation of new neocortical-EC graft-hippocampal circuits.

Transplanted embryonic entorhinal neurons make functional synapses in adult host hippocampus.

Grafts of embryonic entorhinal cortex (EC) or non-entorhinal cortex (NEC) were placed into the hippocampus of adult rats with transection of the perforant paths. Graft-host connectivity was investigated at 4-6 months post-transplantation by recording extracellular evoked responses in hippocampal slice preparations. Electrical stimulation of the grafts evoked excitatory postsynaptic potentials (EPSPs) in the outer molecular layer of the dentate gyrus, and the stratum lacunosum moleculare of CA1, CA3, and elicited population spikes in the granule cell layer and the pyramidal cell layer of CA1, but not CA3. While the latencies and the forms of these evoked response were similar to those in matched control slices from the normal animals, the amplitudes were smaller than normal controls. However, in the slices with NEC grafts, no such responses were recorded when stimulus was applied in similar position in the grafts. The findings suggest that grafted entorhinal neurons make viable synaptic connections with the host hippocampus.