WO2004015102A1

WO2004015102A1 - Olfactory ensheathing cells (oecs) in an extracellular matrix for use in axon regeneration

Info

Publication number: WO2004015102A1
Application number: PCT/GB2003/003470
Authority: WO
Inventors: Geoffrey Raisman; Ying Li
Original assignee: Medical Research Council
Priority date: 2002-08-07
Filing date: 2003-08-07
Publication date: 2004-02-19
Also published as: GB0218316D0; AU2003249092A1

Abstract

The invention relates to a method of inducing axon regeneration in central nervous tissue which has been damaged by a lesion, which method comprises implanting into the lesion a preparation of olfactory ensheathing cells (OECs) which are in an extracellular matrix (ECM) produced by the OECs cells themselves.

Description

OLFACTORY ENSHEATHING CELLS (OECS) IN AN EXTRACELLULAR MATRIX

FOR USE IN AXON REGENERATION

Field of the Invention.

The present invention relates to improved methods of regenerating damaged axoris of the central nervous system (CNS) by transplantation of cells which assist axon regeneration.

Background to the invention.

Nerve cells in the brain and spinal cord communicate with each other by means of myelinated axonal processes, which can be up to a metre or more in length, and which travel through pathways, called white matter tracts, to reach their destinations. The white matter tracts consist of a highly organized cellular substrate, made up of several types of glial cell (astrocytes, oligodendrocytes, which produce myelin, and microglia) . The glial cells are far greater in number than the nerve cells, and are woven into a complex tapestry of almost crystalline regularity. During development, the progressive assembly of this glial cell array provides cues that are essential for the nerve fibres to find their correct pathways.

The nervous system is subject to two unique types of injury: one (typified by spinal cord injury and strokes affecting fibre pathways) in which the axons are severed (axotomy) , and another (typified by multiple sclerosis) in which the axons lose their myelin sheaths. Axotomy leads to the disconnection of nerve cells. Demyelination impairs conduction. Both result in loss of function. After axotomy in the adult central nervous system (CNS) , the cut ends of the axons sprout, but the sprouts are unable to grow back along their original pathways, and the functional loss is permanent. The injury also damages the glial cells and disrupts the regularly aligned glial array of the white matter. The response of the glial cells to damage leads to death of oligodendrocytes, changes the anatomical arrangement of astrocytes (often referred to as a ^Λscar' ) and leads to the production of a matrix of damage-induced molecules.

By contrast, in the peripheral nervous system, a proportion of the axonal sprouts are able to regrow to their original destinations and restore function after axotomy. This favourable outcome depends on the specialized glial (Schwann) cells of the peripheral nervous system responding to the injury by forming a new pathway along which the axons can regenerate. Transplants of Schwann-cell-containing segments of peripheral nerve have been shown to stimulate the growth of cut central nerve fibres, and peripheral nerve grafts are able to restore functional connections from the eye to the brain. But in this situation, the grafts provide a bridge that leads the fibres all the way to their terminal field in the grey matter, without the necessity of their re-entering the white matter tracts of the CNS.

Transplantation of cultured Schwann cells into lesions of white matter tracts of the spinal cord, while stimulating axon growth, does not provide an adequate interface for the growing axons to leave the graft and re-enter their original white matter pathways in the host. The bridge is one-way; it encourages the traffic to get on, but does not allow it to get off again; it is effectively a cul-de-sac. To extend these promising, but incompletely reparative results after Schwann cell transplants, investigators sought a more effective type of glial cell for transplantation. They found it in the nose. The neurosensory cells of the olfactory mucosa are a population of adult neurons that are continually renewed throughout life. The newly formed olfactory nerve fibres leave the olfactory mucosa, pass through the cribriform plate, and form terminals in the glomerular layer of the olfactory bulbs. The remarkable growth potential of the olfactory nerves was shown in a study where the olfactory bulb was removed at birth, and the ingrowing olfactory nerves were seen to enter the adjacent brain area - the neocortex - and form connections there. The olfactory nerves contain not only nerve fibres, but also a special type of glial cell, which later came to be called olfactory ensheathing cells (OECs) . The OECs have a unique arrangement in which they give rise to very fine cytoplasmic processes that enclose huge numbers of unmyelinated olfactory axons. A study in the accessory olfactory (vomeronasal) system showed that the OECs have a special relationship with the olfactory nerve fibres, accompanying them from the olfactory nerve fibre layer all the way to their synaptic terminals in the glomeruli of the olfactory bulb. The ability of OECs to penetrate the surface of the CNS is in contrast to the situation at the entry point of the central branches from the dorsal root ganglia. Here, the Schwann cell ensheathment ceases at the point where the incoming sensory axons meet the central nervous glia, and does not accompany the axons to their terminations in the CNS.

The olfactory ensheathing cell, therefore, became a strong candidate for a reparative cell, superior in effectiveness to Schwann cells. Ramόn-Cueto and Nieto-Sampedro (Exp . Neurol . , 127; 232-244 (1994)) transplanted OECs into the entry zone of cut dorsal root fibres, and showed that this now allowed the regrowing fibres to enter the spinal cord. When cultured OECs were transplanted into complete transection lesions of the thoracic spinal cord, Ramόn-Cueto et al ( J. Neurosci . 18; 3803- 3815 (1998) reported long-distance regeneration of spinal axons. Transplanted into a focal lesion of the rat corticospinal tract, OECs inhibited the profuse axonal branching ( abortive sprouts' ) , and led to a directed elongation of regenerating labelled corticospinal axons not only across the lesion site, but also back into the distal part of the damaged white matter tract. Both these studies noted that, compared with Schwann cells, the transplanted OECs integrated better with the host glia, and migrated further along the host white matter tracts. The transplanted OECs initially expressed the low-affinity neurotrophin receptor p75 (typical of OECs in situ) , and then downregulated it as they remyelinated the regenerating axons with typical peripheral, PO-positive (Schwann type) myelin. When the regenerating axons re-entered the distal part of the corticospinal tract, they became myelinated by oligodendrocytes. This axonal regeneration was accompanied by restoration of a directed paw retrieval function lost after the lesion.

Li et al { The Journal of Neuroscience . 1998, 18; 10514-10524) describe regeneration of adult rat corticospinal axons induced by transplanted olfactory ensheathing cells. The authors describe preparation of these cells by culture of cells from rat olfactory bulbs which are placed in suspension and injected into the site of a lesion. The suspension contains two main types of cells; Schwann-like S cells and A cells, which stain positive for fibronectin.

There is still a need to improve the efficiency of OEC transplant therapy, so that OECs introduced into a lesion of the spinal tract can establish themselves and induce repair.

Summary of the invention.

The present invention provides a method of inducing axon regeneration in central nervous tissue which has been damaged by a lesion, which method comprises implanting into the lesion a preparation of olfactory ensheathing cells (OECs) which are in an extracellular matrix (ECM) produced by the OECs cells themselves .

Description of the Drawings.

Figure 1 shows the extent of the hemisections plotted on the left side, with the medial boundary of the lesions marked. Each line represents a different animal. Scale bar, 1mm. Scale bar, lmm. Figure 2 shows electrophysiological recording of the rhythmic compound action potential from the phrenic nerve. Left hand column: recordings made during spontaneous breathing. Right hand column: recordings after curarisation and 20-50 seconds of asphyxia.

Figure 3 shows faults in the use of the ipsilateral forepaw for climbing. The total fault score for over two climbs (average of 6 weekly tests) for the 14 individual animals in the lesion- alone group and the 22 individual animals in the lesion + transplant group; inset panel compares group means (± SEM) for lesion-alone (LES) , lesion 4- transplant (TRA) and normal, un- operated (N) .

Figure 4 shows the extent of the hemisections, represented as in Fig. 1, in animals which gave a lower climbing fault score (A), the highest fault scores (B) , and where the fault score was reduced by the presence of the transplants (C) . Scale bar, 1mm.

Figure 5 shows schematically the transmission of the supraspinal rhythm and the effect on the transmission of spinal lesions.

Detailed Description of the invention.

The present inventors have found that rather than suspending cells and injecting the suspension at the site of a lesion, the presence of fibronectin-producing "A" cells in an OEC culture allows a matrix of these and Schwann-like "S" cells to be administered directly to a lesion in the CNS.

We have found that the extracellular matrix produced by the OECs themselves provides a viable and useful support for OECs such that they may be transplanted efficiently.

The ECM is secreted by cells and contains molecules such as collagen, fibronectin and elastin in a hydrated glycos- aminoglycan gel. ECM is known to form the scaffold in which cells sit and is also known to provide vital clues for cell migration and proliferation. ECM constituents vary in different tissues. The present invention uses the ECM in which the cells are normally found so it therefore has all the necessary cues for survival and migration of the OECs and supporting regeneration of axons.

This procedure is in contrast to the prior art where researchers have attempted to isolate cells alone or incorporate them into other support media, though this process results in loss of lots of cell specific factors, some of which may not even be known or recognised as important. Thus it is customary in the art to purify cells (i.e. remove their normal ECM) and generate a single cell suspension (by trypsinization or mechanical disruption) . In doing so, vital cell specific components may be lost trypsin breaks ECM molecules down) or will diffuse away after the cells have been injected at a site in a subject.

In contrast, the present invention involves the use of cells which are not disrupted in any way from the matrix, other than by the physical process of recovery from culture (e.g. by scraping the a culture dish) . In the process of the present invention, the cells are recovered without being trypsinized (or dissociated in any other way) . The cells are kept in a semisolid layer/mass with the extracellular matrix with which they are found in after culture. By avoiding going through a 'liquid' stage, all the vital components are physically restrained at the implant site.

The extracellular matrix may be produced by culturing tissue from a source of OECs for a suitable time period to allow the extracellular matrix to be produced. Suitable time periods are from 10 to 20 days, for example from 14 to 17 days. Culture conditions for these cells are well known in the art, and typically include Dulbecco modified Eagle's medium (DMEM) with 5-10% (v/v) fetal calf serum.

Where the cells have an extracellular matrix, they may be prepared simply by growing the cells in culture, scraping the cells from the culture dish, and forming them into a suitable mass for implantation.

In this embodiment, the layer of cultured cells may be folded of formed to provide a multi-layered structure of cells which can be fashioned or further folded into a shape which can be inserted into the site of the lesion.

A layer of cells formed by culture of OECs is about 50 μM thick. In culture, the fibroblast-like cells are flat, and by two weeks in culture they separate from the Schwann-like cells, and form a monolayer under the layer of Schwann-like cells, which are spindle shaped. We have found that such a preparation of cells in culture may be scraped to accumulate a malleable mass of a few (e.g. 2-3) millimetres in diameter, which can be further shaped or cut to fit a transplantation cavity. Several such masses of cells may be brought together to form a larger mass of cells, or used in combination for introduction into a transplant cavity. Typically, the cells may be accumulated to be in a mass of from 1 to 10 mm thick, and with a depth and width within the same range.

The OECs may be obtained from the outer nerve and glomerular layers of an olfactory bulb. See for example, Li et al , supra . Other sources of OECs have been described in WO 01/30982 and include the olfactory mucosa and the lamina propria. These sources may also be used. Olfactory mucosal cells are preferred. Cells may be obtained using surgical methods known as such in the art, for example as described by Li et al , supra, or in WO 01/30982, and references cited therein. Desirably, the OECs are a mixture of Schwann-like cells and fibroblast-like cells.

By "Schwann-like" it is meant OECs which stain positive for the p75 low affinity neurotrophin receptor (Li et al , supra) . A mouse antibody to this is available commercially from Boehringer Mannheim, Germany, and an anti-human p75 antibody may be obtained from Chemicon or generated using the p75 protein as antigen.

S cells can alternatively or also be characterised by the presence of the S100 antigen, which is widely available commercially (e.g. from Sigma Biosciences) .

By "fibroblast-like" it is meant an OEC which show expression of fibronectin in culture. Antibodies to fibronectin are widely available commercially (e.g. Life Technologies or Sigma).

By "mixture" it is meant that at least some of each type of cell is detectable in a sample of the mixture which is to be implanted. It is of course not practical to accurately measure the number of each type of cell, though an acceptable estimate of the proportion of each cell type can be made by immunostaining a sample of the cells prior to implantation. Alternatively, the sample may be subjected to a more quantitative technique such as fluorescent activated cell sorting.

At a minimum, it is desirable that the ratio of Schwann-like to fibroblast-like OECs is in the range of 10:90 to 90:10.

It is preferred that the matrix contains approximately equal numbers of said Schwann-like cells and fibroblast-like cells. In any event, by "approximately equal numbers" it is meant a ratio of the two types of about 50:50, which for practical purposes will be from 40:60 to 60:40 as determined by the objective skilled practitioner. The ratio may be determined by methods such as those discussed above, e.g. immunostaining, or by simple morphology or microscopy. It is not essential to determine the proportion of cells prior to transplantation; using the cell culture methods described herein will generally produce a suitable proportion of both cells types.

The method of the present invention is demonstrated in the accompanying example in a rat animal model. However, the method may be used in other vertebrate animals, including primates and in particular humans.

The OECs used in the method of the invention may be obtained from any suitable vertebrate source. In the case of axon regeneration in humans, the cells are desirably human cells. Human cells may be taken from donor tissue or, where the OECs are readily accessible by simple surgical procedures, they may be obtained from the subject being treated, in order to minimise rejection.

A further source may be from xenografts. There are currently a number of transgenic animals being bred to contain human cell surface markers and/or not to contain cell surface markers which provoke transplant rejection. Such animals may in future provide a source of "universal" donor tissue from which OECs may be obtained.

Cultures of OECs may be created and frozen for future use, or used directly after culture to produce a matrix.

The number of cells which may be implanted at the site of a lesion will depend upon a number of factors, including the size of the lesion and the condition of the subject being treated. The present invention however is an improvement on prior art suggestions of using cell suspensions, in that it allows fewer cells to be used. For example, a suspension of 250,000 cells would typically be required to fill a corticospinal tract lesion of 0.5 mm diameter. But transferred as a matrix, between 1/10 and 1/100 this amount of cells would be needed.

Usually, from 10⁴ to 10⁷ cells may be implanted, though the exact number will depend upon the size and nature of the lesion.

Implantation of the cells will usually be by surgery to expose the site of the lesion so that the cells may be inserted directly. However, the matrix of cells is sufficiently semi- solid such that it may be loaded into a tube and injected.

The treatment may be repeated as often as necessary, and the patient monitored for symptoms of recovery.

The implantation of the cells has been found to allow regrowth of axons in the CNS as described in the accompanying example. In the context of the present invention, the regrowth of axons provided following implantation of the cells will be any level of growth which restores a degree of useful function the patient, e.g. such that the patient regains control over limb movement to at least some extent.

The present invention is illustrated by the following examples.

Spinal cord lesions above the level of the third cervical segment sever the continuity of the bulbospinal respiratory pathways which carry the rhythmic impulses needed to activate the phrenic motoneuron pool lying in the ventral grey matter between the third and fifth cervical segments. Clinically such patients become dependent on continuous artificial respiration. A number of recent studies [Refs. 1-4 below] have put forward evidence that structural and functional repair of the adult rat spinal cord can be achieved by transplantation of glial cells obtained from the primary olfactory pathways, and this has been proposed as a model for clinical application [5] . From a practical point of view, it would be difficult to study the repair of the supraspinal respiratory pathways in animals requiring to be maintained on chronic artificial ventilation due to complete loss of function. We therefore sought partial lesions which would consistently remove a part of the respiratory pathway while sparing sufficient of the supraspinal control mechanism to maintain respiratory function in normal cage behaviour. For this purpose we took advantage of the fact that the bulbospinal respiratory pathways are bilaterally symmetrical: high spinal hemisection abolishes phrenic nerve and diaphragm function on the side of the lesion, but leaves the opposite side functionally intact.

Transplantation of Olfactory Ensheathing Cells (OECs)

Our previous results were obtained with injections of suspensions of cells into lesions of the corticospinal tract, where the functional deficits were assessed in a directed forepaw reaching task [1] . In the present study we used knife cuts to sever the entire spinal grey and white matter on one side at the level of the second cervical segment in adult rats. The larger amounts of tissue destruction are more comparable to those encountered in clinical practice. In addition to respiration, the larger lesions enable us to study a wider range of functions, such as the coordinated movements used in climbing. From a practical point of view, however, the injection of cell suspensions which was adequate to repair small, circumscribed electrolytic lesions of the corticospinal tract, would be highly inefficient for the large hemisections, which are in effect semicircular cuts, open along all their periphery. To permit retention of the transplanted cells in these larger lesions, we have developed a method of transplantation of cells together with the extracellular matrix associated with these cells and obtained by culturing the olfactory ensheathing cells together with a roughly equal number of the specialised fibroblast-like cells with which they are associated in the olfactory nerve layers of the olfactory bulb in situ. This matrix transfer method not only avoids loss of cells during transfer, but also prevents diffusion of the cells away from the transplant site after operation.

Confocal images of 100 μm thick horizontal sections of OECs labelled with an adenoviral GFP construct [6] were taken at 3 days after operation and at 10 days. The images revealed that the celled formed a large and dense meshwork in the lesioned area. At 10 days the cells adopted an elongated shape, aligned rostrocaudally. This shows that there was highly effective retention of transplants of around 500,000 GFP transfected OECs. Comparison of images at 3 and 10 days showed that there is no diminution or diffusion of the transplanted cell mass between these points, by which time the cells are showing the marked alignment response to the local tissue factors which indicates that they have been retained in position long enough for their interaction with the host tissue to be established and their permanent incorporation to be secured.

Immediately after operation all animals showed an unsteady gait and reduced nociceptive responses in the contralateral hind-paw. In both lesion-alone and lesion+transplant animals, all symptoms improved over the first week after operation. This improvement was markedly more rapid in the transplanted animals, but by 2 weeks both groups had reached a level of cage behaviour that was indistinguishable from each other and from that of unoperated rats.

Breathing

A series of 23 hemisected animals was used to investigate the effects of lesions on the supraspinal control of the respiratory rhythm in the ipsilateral diaphragm. Figure 1 shows the extent of the hemisections plotted on the left side, with the medial boundary of the lesions marked. Each line represents a different animal. 14 of these animals had complete hemisections which destroyed the entire half of the spinal cord, reaching at least as far as the midline (Figure IB) . These animals showed an observably asymmetric breathing movement, in which the flaccid hemidiaphragm was pulled to the un-operated side by contraction of the hemidiaphragm of the intact side during the inspiratory phase of breathing. However, when compared with normal intact rats there were no decreases in tidal volume or increases in respiratory rate either at rest or after 5% C0₂ stress, indicating that the asymmetric diaphragm movement moved enough air to maintain the respiratory needs of normal cage behaviour.

In a terminal procedure under anaesthesia, the electrical activity of the phrenic nerves was recorded. All 14 animals with complete hemisection had total unilateral loss of the supraspinal respiratory rhythm in the phrenic nerve on the operated side both during spontaneous breathing and also on a ventilator after a combination of curarisation (to block any peripheral input) and brief asphyxial stress (to maximise the discharges in the descending supraspinal pathways) . The rhythm in 6 representative animals with complete hemisections which include the ipsilateral ventral funiculus is shown in Figure 2, ( ^LESIONS ALONE' ) , which can be compared to two unoperated animals (Figure 2, ^ΛINTACT CONTROLS').

In 9 animals the hemisections were incomplete, i.e. had severed the lateral and dorsal parts of the cord, but had spared the ventral white columns (see Figure 1A, (***) ) . In all these cases, the respiratory rhythm was present in the phrenic nerve of the operated side. This is in agreement with published evidence for a ventrolateral location of the descending bulbospinal respiratory pathways [7-9] .

In a further 8 animals, 500,000 OECs were transplanted into the hemisections. Subsequent histology confirmed that all these hemisections were complete (Figure 1C) , i.e. were equal to, or larger than those shown in Figure IB. At terminal examination, one of these animals did not have a respiratory rhythm in the ipsilateral phrenic nerve. In 7 of these transplanted animals the phrenic nerve showed a clear respiratory rhythm during spontaneous breathing (the rhythm from 6 animals is shown in Figure 2, LESIONS WITH TRANSPLANTS'), and in 5 animals the rhythm was maintained during curarisation and asphyxia.

Climbing

From 2 weeks after operation rats were tested by allowing them to climb twice up an inclined grid at an angle of 15° to the vertical. Climbing the grid is a spontaneous response acquired easily, and requiring neither compulsion nor reward. The climbing test was repeated three times a week for seven successive weeks. Unoperated animals climb readily, with all four paws locating and grasping the bars without fault. In all operated animals, the fore-paws and the hind-paws on the operated side showed varying degrees of difficulty in locating the grid bars. As a result, the fore-paws overshoot the bars, to a depth varying from the wrist to the axilla. The rat senses the overshoot, and the paw is withdrawn, and explores until it makes contact with the bar, which is then grasped as normal, and used as an anchor for the propulsive phase of the climb. In the disadvantaged paws, the propulsive force needed for climbing appeared normal, and the strength of the grasp (assessed by pulling the rats off the grid) was equal on the two sides. To distinguish between lesion-alone and the lesion+transplant groups of animals, one of the three weekly tests was video recorded for each of the 6 successive weeks from weeks 3 to 8 postoperatively. The faults in the forepaw on the operated side were scored by a l/25^th split frame video analysis of all records, with a score of 1 for overshooting through the grid to the level of the wrist, 2 to the elbow, and 3 to the axilla. This method of scoring is objective, based on permanent records, and gives consistent values for different observers.

Over the 6 weeks of the tested period, the individual animals in both groups showed a stable weekly fault score with neither improvement nor deterioration. In Figure 3 we show the results for the 14 lesion-alone rats and the 22 lesion + transplant rats where the subsequent histology indicated that the lesion had reached to the midline. For the lesion-alone group, the average fault score for the six weekly totals was 226±18. The lesion + transplant animals had considerably lower fault scores than the lesion-alone animals, with a mean of 55+7 (Figure 3, Student's t=9.7, P<0.0001). They did not, however, recover to the level of unoperated animals (2+0.2, n=6) . In 5 of the lesion-alone animals where the subsequent histology showed that lesion had spared the dorsal columns, the climbing deficit was less severe (group mean 79+15, t=6.3, P<0.0001) than in the animals with complete hemisections.

Figure 4 shows the extent of the hemisections, represented as in Figure 1, in animals which gave a lower climbing fault score (Figure 4A, score 79±15) . These hemisections spared the region (**) of the dorsal columns and corticospinal tract. Lesions which were complete hemisections (Figure 4B) gave the highest fault scores (226+18) while in transplanted lesioned rats with complete hemisections (Figure 4C) , the fault score was reduced by the presence of the transplants (55+7) .

Discussion

In evaluating functional outcomes, it is important to realise that rat spinal injuries do not produce the same degree of incapacity as human injuries, and also that in both rat and human, major functional improvements occur spontaneously, and can be greatly enhanced by rehabilitative measures, without any reparative interventions in the spinal cord. The transplantation of cells is by no means the only cause of recovery in lesioned animals. All lesioned animals, with or without transplants, improve for the first 10 days after operation. Possible factors contributing to this improvement include reduction of oedema, clearing of debris, vascularisation, sprouting of new connections in denervated areas, and re-programming of existing connections. Our observations show that after the initial recovery period, however, the lesioned animals have persistent deficits in important complex functions, such as breathing and climbing.

Culturing a mixture of OECs and their associated specialised fibroblast-like cells results the production of an extracellular matrix which avoids cell loss during transfer and enables efficient retention of the transplanted cells in large, and their incorporation even in large open lesions. In the case of the return of the supraspinal respiratory rhythm to the phrenic nerve in the transplanted animals, the failure of this rhythm to return in the lesion-alone group of animals, even during asphyxia, indicates that the recovery absolutely depends upon the transplanted OECs. In connectional terms, this recovery could be due to regeneration of severed ipsilateral supraspinal connections across the C2 lesion, and/or to activation of crossed pathways [10,11], enabling the rhythm to gain access the phrenic motoneuron pool at C3-5. This is illustrated schematically in Figure 5. The supraspinal rhythm is conveyed by the descending bulbospinal pathways (left and right arrows, top) through the ventrolateral white matter to the phrenic motoneuron pool (dots) which acts a unit extending from C3 to C5 and transmitting the rhythm to the phrenic nerve where we record it. After a lesion of the descending pathway on one side, the rhythm only reappears in the phrenic nerve of the transplanted animals. This may be mediated by regeneration of the ipsilateral descending pathway (dashes, left) and/or sprouting fibres crossing from the contralateral tract (dashes, bottom) . An overall improvement in hind limb function during climbing has been reported in animals with OECs transplanted into complete mid-thoracic spinal cord transactions [3] . The present study indicates that more precisely quantifiable climbing deficits can be produced and studied in the ipsilateral forepaw after upper cervical hemisections, which leave the animals in overall much better condition, and demonstrate consistent and robust improvement after transplantation. This provides a simpler and reliable experimental model to study the effects of different candidate reparative cell types and molecular interventions [12- 14] .

The histological analysis of the lesions indicates that return of breathing reflects repair of damage to ventrally located tracts, and the return of climbing reflects repair of damage to dorsally located fibre tracts. It seems likely that these beneficial effects of the transplanted OECs are due to regeneration of cut fibres (as reported in other studies, e.g. [1,2]) restoring access to neural information otherwise made unavailable by the lesion. These observations are encouraging for the future use of autografts in human spinal cord injuries.

Methods

Operation

41 adult female inbred AS strain rats were used. The supraspinal muscles were parted in the midline, atlanto- occipital membrane incised, and one half of the upper cervical spinal cord was transected under the operating microscope with a small knife (Fine Science Tools™ No. 10072-12), taking care to avoid damage to the dorsal median blood vessel. This operation has the advantage that there is virtually no bleeding, and no damage to bone or ligament. It can be repaired by skin suture, and causes minimal if any local operative discomfort. Protocol for Extracellular Matrix Transfer

In 22 animals the lesions were filled at the time of operation with around 500,000 of a mixture of approximately equal numbers of Schwann-like and fibroblast-like olfactory ensheathing cells, which were derived from trypsinized tissue taken from the superficial layers of the adult rat olfactory bulb, cultured for 14-17 days and suspended in a matrix of their own production. The tissue was dissected from the outer nerve and glomerular layers of the rat olfactory bulb, trypsinized and plated on to 35 mm dishes. The initial plating density is critical for the cells to produce a sufficiently firm matrix to allow transfer into the lesion site. A good guide is to plate the pooled tissue from 6 bulbs on to 4 dishes. The optimal cell mixture is obtained between 14 and 17 days culture in DMEM-F12 medium including 10% foetal calf serum (Gibco 3133-028), at which time each dish yields about 1.5 million cells of which around 50% are p75 positive Schwann-like cells and 50% fibronectin positive fibroblast-like cells, and the dish becomes completely covered with extracellular matrix around 20 microns thick. The matrix is scraped off the dish with a polythene scraper (Costar 3010™) , gathered into a mass of around 5 mm and cut into 3-4 pieces. Using No 5 watchmaker's forceps (Dumont™) the pieces are lifted out of the dish and transferred directly into the lesion area. Each piece is able to fill a hemisection lesion of around 2 squ mm area, with an edge separation of around 2 mm. This protocol minimises loss of cells during transfer. The efficiency of cell transfer (estimated by the number of cells needed to fill a lesion) is more than 100 times greater than with cell suspensions [1] , but the overall advantage over using a cell suspension is effectively very much greater, since the matrix retains the cells in the lesion site, preventing loss by diffusion, and obviating the need for introducing any exogenous matrix materials. Electrophysioloqical Recording of Respiratory Rhythm in the Phrenic Nerve [15]

Under deep anaesthesia, controlled by continuous monitoring of blood pressure, the rats were endotracheally intubated, and the phrenic nerve on the operated side was exposed, immersed in a pool of warm paraffin oil, cut distally and placed on bipolar tungsten electrodes. The recordings were amplified (0.5 to 2 K) , filtered (50 to 3000 Hz) and fed into a "leaky" RC integrator with a time constant of 50 ms . After recording the activity during spontaneous breathing, the animals were artificially ventilated with a mixture of room air (50%) and oxygen (50%) at a rate of 40-60/min (tidal volume 2-4 ml) and paralyzed with a neuromuscular blocking agent (Flaxedil®, gallamine triethiodide i.v. 10 mg/kg, to block any peripheral input) and the brain stem respiratory centres provoked to maximal output by temporarily stopping the ventilator (20 to 50 seconds) .

Histology

After the six weeks of serial functional testing for climbing and terminal electrophysiology of the phrenic nerves, the rats were perfused with phosphate buffered saline, and the extent of the damage in each animal was assessed by reconstruction from 20 μm thick serial longitudinal cryostat sections taken through the entire width of the spinal cord through the level of the lesions/transplants, and stained with aqueous thionin (for cell bodies) or immunostained for neurofilament (for axons; Chemicon 1:500). Hemisection produces consistent lesions, with minimal damage to surrounding intact tissue, and the extent of the lesions in each individual can be accurately assessed by serial section histology. The hemisections, even those transecting the central canal and reaching beyond the midline, healed with minimal scarring, and no cyst formation or persistent macrophage invasion.

Re erences

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Claims

1. A method of inducing axon regeneration in central nervous tissue which has been damaged by a lesion, which method comprises implanting into the lesion a preparation of olfactory ensheathing cells (OECs) which are in an extracellular matrix (ECM) produced by the OECs cells themselves.

2. The method of claim 1 wherein the OECs comprise a mixture of Schwann-like cells and fibroblast-like cells.

3. The method of claim 2 wherein the mixture comprises approximately equal numbers of said Schwann-like cells and fibroblast-like cells.

4. The method of claim 1 or 2 wherein said cells have been prepared by culturing tissue from the outer nerve and glomerular layers of an olfactory bulb for 10 to 20 days.

5. The method of claim 1 wherein from 10⁴ to 10⁶ cells are implanted.

6. In a method of inducing axon regeneration in central nervous tissue which has been damaged by a lesion by implanting into the lesion a preparation of olfactory ensheathing cells (OECs) , the improvement comprising providing said preparation in the a form in which said preparation of olfactory ensheathing cells (OECs) are in an extracellular matrix (ECM) produced by the OECs cells themselves.

7. A preparation of olfactory ensheathing cells (OECs) which which are in an extracellular matrix (ECM) produced by the OECs cells themselves for use in a method of treatment, said treatment inducing axon regeneration of central nervous tissue which has been damaged by a lesion.

8. A preparation for use according to claim 7 wherein the OECs comprise a mixture of Schwann-like cells and fibroblast-like cells.

9. A preparation for use according to claim 8 wherein the mixture comprises approximately equal numbers of said Schwann- like cells and fibroblast-like cells.

10. A preparation for use according to any one of claims 7 to 9 wherein said cells have been prepared by culturing tissue from the outer nerve and glomerular layers of an olfactory bulb for 10 to 20 days.

11. A preparation for use according any one of claims 7 to 10 wherein from 10⁴ to 10⁶ cells are implanted.