WO1999040924A2

WO1999040924A2 - Treatment of neurodegeneration

Info

Publication number: WO1999040924A2
Application number: PCT/IB1999/000246
Authority: WO
Inventors: José LOPEZ BARNEO; José Angel ARMENGOL BUTRON DE MUGICA; Rafael Jesús MONTORO LASECA; Emilio Fernandez Espejo
Original assignee: University Of Seville
Priority date: 1998-02-11
Filing date: 1999-02-11
Publication date: 1999-08-19
Also published as: ES2145694B1; WO1999040924A3; ES2145694A1

Abstract

An isolated aggregate of hypoxia sensitive cells that release one or more neurotransmitters, neuromodulators or enzymes is provided characterised in that the aggregate is dimensioned such as to be capable of surviving, preferably for 3 months or more, in a host brain suffering from neurodegeneration or cerebrovascular disorder while substantially reversing some or all of the symptoms of that neurodegeneration or cerebrovascular disorder.

Description

TREATMENT OF NEURODEGENERATION.

This invention provides a treatment for neurodegeneration or cerebrovascular disorders, particularly providing a procedure for the delivery of neurotransmitters, neuromodulators or enzymes by the utilisation of hypoxia-sensitive cells in the form of aggregates of such cells, for example aggregates of cells of the carotid body. Preferably the aggregates are in a form that has been subjected to a slight enzyme treatment which does not separate the tissue into isolated cells. Thus the aggregates of tissue so provided may have been treated enzymatically but have not dispersed mechanically. In the delivery procedure the cells secrete neurotransmitters, neuromodulators or enzymes and have utility in the treatment of neurodegenerative diseases arising from the deficit of certain molecules, particularly molecules of a neurotransmitter, neuromodulator or enzyme which would be then released by the transplanted cells of the invention, eg. secreted by an implant of the tissue.

This release or secretion has use in reversing some of the deficits of the disease and achieves a much better recovery than that achieved so far by means of pharmacological administration of these molecules or their precursors. Also in cerebrovascular diseases, in which a loss of part of the cerebral parenchyma has been caused, the cellular implant could assist a substantial recovery of the remaining neurones and optionally replaces some of the lost synaptic circuits. Further provided are preparations comprising hypoxia sensitive cells in the form of cell aggregates, as are methods for the production of such preparations and methods for carrying out medical procedures using such preparations.

STATE OF THE ART Neural or paraneural cells which secrete neurotransmitters, neuromodulators or enzymes have been used for the treatment of degenerative diseases, and are an alternative to pharmacological therapy. This use has been carried out at the experimental level, though so far with limited success in clinical trials.

In the late nineteen seventies and early nineteen eighties beneficial effects were seen with intrastriatal transplants of adrenal medulla, which include dopamine secreting chrornaffin cells, in models of experimental Parkinson's disease in rats (Perlow et al., 1979; Freed et al., 1981). In the nineteen eighties the therapeutic effect of paraneural autotransplants of adrenal medulla was investigated in patients with Parkinson's disease. These transplants demonstrated little functionality and early rejection (Backhand et al., 1985; Madrazo et al., 1987; Lindvall et al, 1987; Peterson et al., 1988). In the early nineteen eighties, the beneficial effect of transplants of neural cells obtained from foetal substantia nigra was observed using an experimental Parkinson's disease rodent model (Bjorklund et al., 1982; Brundin et al., 1987). The functionality and survival of these transplants was better than for transplants of adrenal chrornaffin cells but their use is very limited in certain countries owing to ethical or legal constraints (Freed et al., 1992; Kordower et al., 1995). As a result tests are being carried out at clinical level using xenotransplants of foetal cells of non- human substantia nigra, for example of pig, with promising results.

At present the use of, inter alia, the following secreting cells is also being tried for transplants in treatment of Parkinson's disease: a) dopamine secreting cells derived from human neuroblastoma (lines SK-N-SH, CHP126, BN69 and LAN5); b) multipotent cells genetically modified to produce tyrosine hydroxylase or dopamine (lines HLB5 C17-2, CSM14.1); c) co-transplants of non-human foetal neural cells and cells secreting growth factors (NGF, BDNF, GDNF) and d) co-transplants of non- human foetal neural cells and Sertoli cells which reduce the immune response of the receptor tissue. All these studies are in the pre-clinical experimental phase (Bottenstein, 1981; Gage, 1987; Yurek et al., 1989; Yoshimoto et al., 1995; Sanberg et al., 1996; Martinez-Serrano and Bjorklund, 1997). Although multipotent cells have demonstrated limited functionality, this line of research based on their use is of great interest, while cells which secrete growth factors, especially GDNF, substantially improve the functionality of transplanted doparninergic cells.

In Alzheimer's disease, another neurodegenerative condition, the models used have first provided lesion of the basal nuclei of Meynert and then implants have been made using the zone of the basal ganglia from embryos in the neocortex of animals. Although these implants initially, i.e. in the first 4 weeks, emit prolongations, they are not maintained for longer than 10 weeks (Dunnett et al., 1986). The survival of such transplants improves somewhat with the application of cyclosporin A in the following two weeks (Howard et al., 1988). The evolution of these implants in other zones such as the lateral ventricle or the subarachnoid space has been histologically compared and no great differences exist (Kyoshima et al., 1992). These subarachnoid space implants achieve some improvement even when they do not project beyond the cortex, so it is considered that factors which diffuse from them are responsible for this improvement (Kyoshima, 1993).

Another experimental model for Alzheimer's disease has been the lesion of the neocortex, for example with kainic acid, which causes degeneration of the cholinergic cells of the basal ganglia and nuclei which project beyond that zone of the neocortex. If after the lesion a cellular suspension of embryonic cortical tissue is implanted, degeneration of the basal ganglia and nuclei is prevented, which now project beyond the transplanted cells (Sofroniew et al., 1986). As one of the main problems is the duration of the transplant, biodegradable microspheres have been tried which release nerve growth factor for up to 5 weeks, which may assist with the maintenance of the transplant (Camarata et al., 1992). This release has also been achieved from animal cells genetically modified to secrete nerve growth factor which prevents the degeneration of cholinergic cells (Tuszynski et al., 1996). In Huntingdon's degenerative disease, an animal model has also been used consisting of the lesion of the striatum, which gives rise to hyperactivity of the ariimal and grave deficits in learning. The implant in the injured striatum of a cellular suspension of neurones of embryo striatum leads to an improvement in the symptomatology of the animal and a recovery of the enzymatic activity of the neurones affected by the lesion (Isacson et al., 1985, 1986). The study of these implants with light and electron microscopy reveals an organisation similar to the normal caudate, although with a smaller number of synapses within the transplant (Difiglia et al., 1988, Zhou et al., 1989). These implants also partly prevent lesioning which is caused by injection of kainic or quinolinic acid in the striatum (Tulipan et al., 1988). This preventive effect is also obtained with the application of MK801 (Giordano et al., 1990) or fibroblasts secreting nerve growth factor (Schumacher et al., 1991; Frim et al., 1993), before the lesion is performed.

In primates implants have been made with neurones of the striatum of rat foetuses, accompanying immuno-suppression of the host, but the survival of the transplant is not very great (Isacson et al., 1989). In models with primates, nuclear magnetic resonance has proved to be a useful method for locating both the area of the lesion and the later implant (Simmons et al., 1994). Transplants have also been made with neurones of the striatum of human foetuses in rats, obtaining a good survival of the transplant (Pundt et al., 1996; Grasbon-Frodl et al., 1996). The recovery of the symptomatology is correlated with the number of neurones of the implant which are capable of expressing typical markers of the neurones of the striatum such as phosphoprotein DARPP-32 (Nakao et al., 1996), and implants from younger embryos are better (Flicker et al., 1997).

Most relevantly, Bing et al (1988) reported transplant of dispersed isolated carotid body glomus cells as implants in adult rat striatum. Although they suggest that such implants might be useful, it was found that they only achieved 50% compensation in motor assymetries and that most transplanted cells died within 4 weeks.

The present inventors have now provided preparations of aggregates of hypoxia sensitive cells, such as carotid body cells, which substantially abolish such assymetries and which provide high graft cell survival rates even after 3 months.

GENERAL DESCRIPTION OF THE INVENTION

The present invention provides the use of hypoxia-sensitive cells in aggregate form which release, e.g. secrete neurotransmitters, neuromodulators or enzymes. The term aggregate form refers to undispersed cells in which the intercellular ratio is preferably maintained. Thus such aggregates, preferably in the form of donor tissue, particularly autologous donor tissue, are not enzymically treated or have only been subject to slight enzymatic treatment without mechanical dispersal so that the hypoxia-sensitive cells of the tissue are implanted with little damage while still being surrounded by a certain amount of supporting tissue. Particularly preferred tissue in the form of cell aggregates of the invention include glia or glia-like support cells, such as the carotid body type II cells.

Transplants with the cellular aggregates of the present invention are of clinical use for the therapy of neurodegenerative or cerebrovascular diseases. In these diseases, the deficit of some molecules such as neurotransmitters, growth factors and enzymes, may be compensated by release/secretion from the implanted cells. In conserving their intercellular relations, the cells of the aggregates can survive a lot longer than isolated cells, e.g. up to 4 months in our trials, while the present enzymatic treatment breaks up the conjunctive structures which isolate the cells in the original tissue, facilitating the egress of the substances secreted and also their new connection with the neurones of the tissue it is intended to repair. The fact that cells are used which secrete substances in response to hypoxia has the advantage of making use of the partial low pressure of oxygen, which when these cells are transplanted in the host brain they will find as a stimulus for the formation of neurotransmitters and their later secretion.

Thus in a first aspect of the present invention there is provided an isolated aggregate of hypoxia sensitive cells that release one or more neurotransmitters, neuromodulators or enzymes characterised in that the aggregate is dimensioned such as to be capable of surviving, preferably for 3 months or more, more preferably for 1 year or more, in a host brain suffering from neurodegeneration or cerebrovascular disorder while substantially reversing some or all of the symptoms of that neurodegeneration or cerebrovascular disorder.

Preferably the aggregate comprises a sectioned portion of paraneural tissue of a donor animal, particularly being characterised in that it comprises a sectioned portion of a carotid body. Most preferably the aggregate is prepared with autologous tissue, ie. with tissue that has been taken from a patient that is intended to receive the aggregate as an autologous implant.

More preferably the aggregate comprises sections of from 0.1mm to 0.5mm smallest dimension, still more preferably being sections of from 0.1mm to 0.5mm diameter, particularly being from 0.2mm to 0.3mm diameter and/or smallest dimension. Preferably the aggregates of the invention are cut in the form of blocks, eg. cubes of tissue prior to enzyme treatment.

The total number of cells implanted in a treatment may be for example from 100 to 50,000 cells, depending upon the species being treated. Thus while an individual aggregate may contain, for example, from 100 to 1000 cells, several such aggregates may be implanted.

Preferred aggregates are characterised in that conjunctive structures which isolate cells in the donor tissue have been broken up. Preferably the structures have been enzymatically digested and/or degraded, more preferably using one or more proteases, eg. such as collagenase and trypsin. More preferably the tissue is also treated with DNAase.

The aggregates of the invention and the implants comprising them are preferably such that they are capable of reversing sensorimotor defects in a host suffering from dopaminergic insufficiency in the caudate putamen, striatium and/or substantia nigra, particularly in Parkinson's disease and related states. They are more preferably characterised in that they are capable of substantially reducing tremor, akinesia and/or rigidity in a host suffering from dopaminergic insufficiency in the caudate putamen, striatum and/or substantia nigra. Most conveniently, the aggregates of the invention comprise a section of a paraneural tissue, preferably a carotid body, ie. derived from a host animal. The animal is preferably the same animal as that which is to receive the aggregate, eg. in the form of an autologous implant. Preferably the tissue is cleaned to remove surrounding adipose and connective tissue prior to enzymic treatment. For use in therapy, the prepared aggregate makes up all or part of a cellular implant suitable for the treatment of neurodegeneration or a cerebrovascular disorder.

A second aspect of the present invention provides the use of an isolated aggregate of hypoxia sensitive cells as described for the first aspect for the preparation of a medicament for the treatment of neurodegeneration or a cerebrovascular disorder, the medicament preferably being a cellular implant, eg. for the treatment of Parkinsonism or Alzheimer's disease.

A third aspect of the present invention provides a method of preparing an implant for use in treating neurodegeneration or cerebrovascular disorders comprising sectioning a tissue comprising hypoxia sensitive cells that release one or more neurotransmitter, neuromodulator or enzymes into pieces, ie. cellular aggregates, without dispersing the cells. Preferably the method treats the sectioned tissue with an enzymic solution such as to increase egress of neurotransmitters, neuromodulators or enzymes. The enzymatic solution comprises proteolytic enzymes. The enzymatic solution preferably degrades or digests connective structures sufficient to facilitates new connection of implant cells with those of a host tissue which is to be treated without separating the cells of the aggregate. Most preferably the enzymatic treatment is with collagenase and trypsin the concentration and time of action of these being insufficient to separate the cells from the aggregate. Preferably the solution further comprises DNAase.

In a preferred embodiment of the third aspect of the invention the enzyme treatment is carried out with a calcium and magnesium free Tyrode solution containing collagenase (about 1 mg/ml), trypsin (about 1 mg/ml) and DNAase (about 0.5 mg/ml). The time of exposure to enzymatic solution will depend upon the activity of that solution. At ambient temperature the treatment will typically last from 5 to 60 minutes for the preferred solution referred to above.

It will be realised that the aggregate is preferably washed to remove enzymes of the treatment solution after treatment to digest or degrade connective structures. Preferably the aggregate is centrifuged and resuspended in physiologically acceptable medium to remove enzymes, eg. a Tyrode solution.

In a fourth aspect of the invention there is provided a method of treating a patient suffering from neurodegeneration or cerebrovascular disorder characterised in that it comprises implanting an aggregate of hypoxia sensitive cells, these cells releasing neurotransmitters, neuromodulators and/or enzymes, into regions of neurodegeneration or cerebrovascular disorder. The aggregate is preferably as described above for the first aspect. The number of such aggregates implanted will depend on the size and species of the patient to be treated. Thus, for small animals, such as rats, cats etc, one aggregate of 20% to 25% of the carotid body glomus may suffice whereas for large animals such as primates and humans, more such aggregates may be required in order to balance the need for sufficient cells with the preferred size of sectioned material. Where the donor is a large animal or human then the number of aggregates making up the implant will necessarily be increased.

Aggregates of the invention are preferably implanted by injecting a physiologically acceptable carrier in which they are floating or suspended into the area to be treated. For primate or human treatment several, for example 2 to 20, aggregates of 0.2mm may be needed to be implanted along a syringe tract within tissue to be treated. More aggregates may be required dependent upon the severity of the condition being treated. Number of aggregates used will of course correspond with aggregate size within the range in which aggregates are viable long term, i.e. for 3 months or more in the host brain.

A fifth aspect of the present invention provides a composition comprising a suspension of from 1 to 100 aggregates of the invention in a physiologically acceptable medium. Preferably the medium is an aqueous medium, more preferably being a Tyrode medium.

A sixth aspect of the present invention provides a method for the delivery of neurotransmitters, neuromodulators or enzymes, characterised in that it uses an aggregate of hypoxia-sensitive cells of the invention that secrete the neurotransmitters, neuromodulators or enzymes . A seventh aspect of the invention provides a set of materials and instruments, for preparing aggregates of the invention by the method of the invention, the set preferably being in kit form, comprising

(i) a slicing means dimensioned such that it produces one or more blocks of tissue of from 0.1mm to 0.5mm smallest dimension from a section of donor tissue and (ii) a composition containing enzymatic components capable of degrading or digesting connective structures in the blocks of donor tissue when in solution form.

Preferably the slicing means comprises a number of cutting elements that are spaced such as to define tissue block dimensions. Still more preferably, the tissue is placed on or in a reception chamber or zone of the slicing means, in which the cutting elements are caused to engage the tissue and section it into blocks of the size required for implant.

Preferably the composition is freeze dried, requiring the addition of fresh liquid, preferably an aqueous liquid such as distilled or deionized water, prior to use. Preferably the composition is as particularly described in the third aspect of the invention, eg. comprising collagenase and trypsin and more preferably also DNAase. The set or kit preferably further comprises one or more of (iii) a vessel for incubating tissue in the enzymatic solution, (iv) a marker device for indicating when the digestion is completed, (v) components suitable in which to suspend the block of enzymatically treated tissue for removal of enzymes and/or for transplant into a host brain and (vi) an implantation device for receipt of the suspended tissue blocks, ie. the aggregates of the invention, and delivery of these into said brain.

Preferably the marker device produces a visible colour change, or provides a contrast against which the completion of change in the tissue is more readily seen, such that incubation in the enzymatic solution may be controlled reproducibly. Preferably the components (v) are those used to clean and suspend the tissue blocks as described in the third aspect. Conveniently this will be a Tyrode solution. Device (vi) is preferably a syringe of with a needle of gauge suitable to allow passage of blocks of tissue of up to 0.5mm, and possibly larger, dimension.

DETAILED DESCRIPTION

The present invention will now be described further by reference to the following experiment presented below. The experiment together with the attached figures illustrates the invention in non-limiting fashion and is based on the use of cellular aggregates of the carotid body for the treatment of experimental Parkinson's disease in rodents. It must be understood the invention may be applied to other animals, such as primates and human beings, and to other degenerative or cerebrovascular patterns.

FIGURES:

Figure 1. Amperimetric measurements of the dopamine signal in animals with lesions and animals having implants of the invention. A: shows two readings taken in the same animal in normal (N) and denervated (D) striatum. B: compares the average of 6 measurements in normal striata (white rectangle) with the average of 7 measurements taken in denervated striata (black rectangle). C: shows two recordings made in another animal in its normal (N) striatum and in its reinnervated striatum (R), three months after the transplant. D:. Compares the average of 5 measurements made in a normal striatum (white rectangle) and 5 measurements made in a reinnervated striatum (black rectangle). In A and C, the lines with a K indicate the time of application of a solution of 66mM KC1. In B and D the bars indicate standard deviation and SD on the y-axis signifies a secretion of dopamine measured in picoamperes.

For amperometry the animals were decapitated under anaesthetic and the brain was cut coronally in cold Krebs-Ringer solution in slices of 150-200 Dm. For the amperometric measurements, the slices were placed in a reading chamber perfused with a solution of (values in mM): 150 NaCl, 2.7 KC1, 2.5 CaCl₂, 1 MgCl₂ and 10 Hepes (pH=7.4). To induce the release of dopamine it was replaced with another similar solution but with 66 mM KC1 and 84 mM NaCl. The dopamine was recorded using a carbon fibre electrode of 12 Dm diameter, connected to a current- voltage converter and polarised at a constant voltage of +650 mV. The amperometric signals were filtered at 50-100 Hz and recorded.

Figure 2. Morphological characteristics of transplants of carotid body three months after implantation. The coronal sections through the striatum show the shape (A, B) and organisation (C, D) of the transplants. Glomic cells both isolated and in glomeruli (arrow head) are observed, with evident neuronal morphology, presenting long dendrites (arrows) Calibration: A, 1 mm; B, 500 Dm, C-D 15 Dm.

For immunocytochemistry the rats were perfused with 150 ml of phosphate buffer saline solution (PBS) followed by paraformaldehyde at 4% in a phosphate buffer (PB) 0.1 M (pH 7.2-7.4). After extraction the brain was fixed with paraformaldehyde in a phosphate buffer (PBS) 0. 1 M (pH 7.2-7.4) and was immersed in 30% saccharose with PB until it was moistened. Coronal sections of 30 Dm thick were made with a freezing microtome and were incubated in PBS/0.2% gelatine/0.1% Triton X-100 (PBS-G-T) with normal goat serum (vector), for 1 h to block specific binding sites. These sections were then incubated overnight with polyclonal anti-TH antibodies (1:30000) in PBS-G-T. After washing with PBS-T, the sections were incubated in biotinylated rabbit antibody (1:200) and then incubated with an ABC kit (L100 Vector) for 1 h and the bound antibody disclosed using 3.3'-diaminobenzidine. After washing in PBS, the sections were mounted on gelatinised slides, dehydrated and covered.

Figure 3. Evolution in time of rotation and sensorimotor orientation in control rats (open circles), Parkinsonian rats with transplant of carotid body (filled squares) and Parkinsonian rats with sham transplants (open triangles). Rotation was evaluated by spontaneous net rotation in the open field test (A), and the rotation induced by amphetamine (B). Sensorimotor orientation was evaluated by the whisker touch test (C) and net thigmotactic locomotion in the open field (D). Averages ± EEM * p < 0.05, ** p < 0.01 with regard to rats with sham transplants; # p < 0.05, ## p < 0.01 with regard to Parkinsonian rats with transplant of carotid body or sham transplant. Abbreviations of the figure: RE: spontaneous rotation; RA: rotation induced by amphetamine; WT: whisker touch test;; LT: thigmotactic locomotion; rtn: net rotation; rt: number of rotations in 60 rnin; lat,: latency (s); tn: net thigmotaxia; cl: contralateral; il: ipsilateral; t: transplant; el: evolution in time; Pre: seven days before the lesion; Les: seven days after the lesion of substantia nigra; lOd, lm and 3m: ten days, 1 month and 3 months after the transplant. The arrow indicates the time of the transplant (ten days after the lesion of the substantia nigra). Net rotation in the open field (1 x 1 m) is defined as the percentage of 360 D turns towards the side of the lesion or ipsilaterai minus the contralateral (10 min test). Amphetamine was injected at 5 mg/kg IP, and induces ipsilaterai rotations, which are quantified from 30 to 90 min following the injection. The whisker touch test consisted of bringing a stick nearer on the right side of the animal until the vibrissae are moved, and measuring the latency of response to the approach of the stick (maximum 25 s). Net thigmotactic locomotion was measured in the open field, being defined as the percentage of the time the animal spends ninning around the enclosure sticking to the walls (ipsilaterai minus contralateral time).

EXAMPLE 1:

For experimentation the model of Parkinson's disease in rats was used, based on the unilateral lesion of substantia nigra by means of a toxic substance, 6- hydroxydopamine, which selectively destroys dopaminergic cells. Rats with a lesion of more than 90% of substantia igra present symptomatology which is similar to Parkinson's disease in humans: i.e. akinesia, rigidity, lateralisation in movements (rotation in the rat) and sensorimotor deficit. These functional defects may be evaluated with an appropriate battery of tests. Furthermore, lateralisation in movements may be exacerbated by the injection of amphetamine (5 mg/kg), which induces intense "ipsilaterai rotation" (towards the side of the lesion) the frequency of which indicates a lesion higher than 90% if greater than 360 turns per hour.

Male Wistar rats (275-325g) were housed at regulated temperature (22DC±1 DC) in a 12 hour light-dark cycle (lights on 08:00 hours). Food and water were available ad libitum. Thirty minutes before 6-hydroxydopamine (6-OHDA, RBI) lesion , rats were injected with desipramine (15mg/kg i.p.) to protect noradrenergic terminals from 6-OHDA toxicity. Rats were anaesthetized with chloral hydrate (450mg/kg i.p.) and placed in a Kopf stereotaxic apparatus with the incisor bar set at 3.3 mm below the interaural line. Saline solution (1 Dl) containing 6-OHDA (4Dg/Dl) and 0.2% ascorbic acid was injected over 15 minutes with ablunted 30-gauge cannula at the following coordinates with respect to bregma: AP=-5.3, L=-2.3, and V=-8.2. The cannula was left in place for 1 minute after injection. After surgery, rats were injected with penicillin (100,000 I.U. i.m.) Control rats followed the same protocol except that a 6-OHDA free solution (0.9% NaCl with 0.2% ascorbic acid) was injected. For each Parkinsonian rat a section of the carotid body of a rat was implanted in the denervated striatum (coordinates AP = 0.2, L = -3, V = -5.5 with respect to bregma) ten days after the unilateral lesion of the substantia nigra by means of 1 Dl of 6-OHDA (4Dg/Dl; coordinates AP = -5.3, L = -2.3, V = -8.2). The section of carotid body (1/4 to 1/5 of the glomus; 400 to 600 glomic cells) had been lightly enzymatically treated without final mechanical dispersal of the tissue. This involved incubation for 20 minutes in Tyrode solution without calcium or magnesium, with collagenase (1 mg/ml), trypsin (1 mg/ml) and DNAase (0.5 mg/ml).

Thus, for the graft preparation procedure, carotid body cell aggregates were obtained from isogenic male rats (275-325g) under anaesthesia. Carotid bifunctions were removed after neck incision, and carotid bodies were isolated , cleaned of surrounding adipose tissue, and trimmed into pieces of approximately 20% to 25% of the whole carotid body. The estimated number of transplanted glomus cells varied between 400 and 600. The tissue was incubated for 20 minutes in a Ca²⁺ and Mg²⁺ free Tyrode solution with collagenase (1 mg/ml), trypsin (1 mg/ml) and DNAase (0.5mg/ml) as stated above. Cell aggregates were centrifuged at 800g for 5 minutes and resuspended in 5ml of normal Tyrode solution to remove the enzymes. The fragments of carotid artery used for sham grafts were prepared following the same procedure.

Ten days after substantia nigra lesion Parkinsonian animals were anaesthetized and placed into a Kopf stereotaxic apparatus. A burr hole was drilled over the denervated stiatum and a blunted 23-gauge cannula, connected to a 2ml Hamilton syringe was lowered to the injection site (coordinates: AP=+0.2, L=-3, and V=-5.5). Tyrode solution (2ml) containing a cell aggregate of carotid body or carotid artery was injected over 5 minutes. After surgery, rats were injected with penicillin (100,000 I.U. i.m.). Ten, thirty and ninety days after the transplant, the functionality and efficacy of the graft was evaluated from a neurochemical, morphological and behavioural viewpoint. The neurochemical study was based on amperometric readings, in slices of brain tissue, of the intrastriatal levels of dopamine in slices of tissue. The morphological studies were of an immunocytochemical nature, by the use of stains and antibodies for tyrosine hydroxylase, which indicate the presence or absence of dopamine producing cells. The behavioural evaluation was made by means of an appropriate battery of tests which made it possible to evaluate the motor and sensorimotor responses of the animal. The functional results were excellent: the amperimetric readings, which detected a fall greater than 90% of dopamine in the denervated striaturn without the transplant, revealed a recovery of up to 65% of the dopamine levels in the striatum. with a transplant of three months' evolution (figure 1). The morphological studies showed the presence of transplants with glomic cells positive to tyrosine hydroxylase, associated in glomeruli and which after three months presented long fibres which went from the transplant to the striatum, which gave the cells a neuronal appearance (figure 2). The fibres presented abundant varicosities in a regular way along their run, suggesting the establishment of dopaminergic synapses en passant, in the same way as axons, do from the substantia nigra in the normal striatum. All the above was correlated with a very good behavioural recovery: a) the rotation, both spontaneous and induced by amphetamine, disappeared during the first month, b) the sensorimotor reflexes were recovered after three months (figure 3).

The application of this method to primates achieves similar success when an appropriately increased number of aggregates, e.g. 2 to 10, is used.

Claims

CLAIMS.

1. An isolated aggregate of hypoxia sensitive cells that release one or more neurotransmitters, neuromodulators or enzymes characterised in that the aggregate is dimensioned such as to be capable of surviving in a host brain suffering from neurodegeneration or cerebrovascular disorder while substantially reversing some or all of the symptoms of that neurodegeneration or cerebrovascular disorder.

2. An aggregate as claimed in claim 1 characterised in that it is capable of surviving for 3 months or more in the host brain while substantially reversing some or all of the symptoms of that neurodegeneration or cerebrovascular disorder.

3. An isolated aggregate of hypoxia sensitive cells characterised in that it comprises a sectioned portion of paraneural tissue of a donor animal or human.

4. An isolated aggregate as claimed in Claim 1, 2 or 3 characterised in that it comprises a sectioned portion of a carotid body.

5. An isolated aggregate as claimed in any one of Claims 1 to 4 characterised in that it comprises sections of from 0.1mm to 0.5mm smallest dimension.

6. An isolated aggregate as claimed in Claim any one of Claims 1 to 5 characterised in that it comprises sections of from 0.1mm to 0.5mm diameter.

7. An isolated aggregate as claimed in any one of Claims 1 to 6 characterised in that it is of from 0.2mm to 0.3mm diameter and/or smallest dimension.

8. An isolated aggregate as claimed in any one of the preceding claims characterised in that it comprises from 100 to 10,000 cells.

9. An isolated aggregate as claimed in any one of the preceding claims characterised in that conjunctive structures which isolate cells in the donor tissue have been broken up.

10. An isolated aggregate as claimed in Claim 9 characterised in that the structures have been enzymatically digested and/or degraded.

11. An isolated aggregate as claimed in Claim 9 or Claim 10 characterised in that the structures have been enzymatically digested and/or degraded using one or more proteases.

12. An isolated aggregate as claimed in Claim 11 characterised in that the conjunctive structures have been broken up by treatment with collagenase and trypsin.

13. An isolated aggregate as claimed in any one of the preceding claims characterised in that the tissue has also been treated with DNAase.

14. An isolated aggregate as claimed in any one of the preceding claims characterised in that it is capable of reversing sensorimotor defects in a host suffering from dopaminergic insufficiency in the caudate putamen, striatium and/or substantia nigra.

15. An isolated aggregate as claimed in any one of the preceding claims characterised in that it is capable of substantially reducing tremor, akinesia and/or rigidity in a host suffering from dopaminergic insufficiency in the caudate putamen, striatum and/or substantia nigra.

16. An isolated aggregate as claimed in any one of the preceding claims characterised in that it is a section of a carotid body.

17. An isolated aggregate as claimed in any one of Claims 3 to 16 characterised in that it has been cleaned to remove surrounding adipose and connective tissue.

18. A cellular implant suitable for the treatment of neurodegeneration or a cerebrovascular disorder characterised in that it comprises an aggregate as claimed in any one of Claims 1 to 17.

19. Use of an isolated aggregate of hypoxia sensitive cells as claimed in any one of the Claims 1 to 17 for the preparation of a medicament for the treatment of neurodegeneration or a cerebrovascular disorder.

20. Use as claimed in Claim 19 characterised in that the medicament is an implant.

21. Use as claimed in Claim 19 or Claim 20 characterised in that the medicament or implant is for treatment of Parkinsonism or Alzheimer's disease.

22. A method of preparing an implant for use in treating neurodegeneration or cerebrovascular disorders comprising sectioning a tissue comprising hypoxia sensitive cells that release one or more neurotransmitters, neuromodulators or enzymes into aggregates without dispersing the cells.

23. A method as claimed in Claim 22 characterised in that it treats the sectioned tissue with an enzymatic solution such as to increase egress of neurotransmitters, neuromodulators or enzymes.

24. A method as claimed in Claim 23 characterised in that the enzymatic solution comprises proteolytic enzymes.

25. A method as claimed in Claim 23 or 24 characterised in that the enzymatic treatment degrades or digests connective structures sufficient to facilitates new connection of implant cells with those of a host tissue which is to be treated without separating the cells of the aggregate.

26. A method as claimed in Claim 25 characterised in that the enzymatic treatment is with collagenase and trypsin, the concentration and time of action of these being insufficient to separate the cells from the aggregate.

27. A method as claimed in any one of Claims 23 to 26 characterised in that the solution further comprises DNAase.

28. A method as claimed in any one of Claims 23 to 27 characterised in that the enzyme treatment is carried out with a calcium and magnesium free Tyrode solution containing collagenase (1 mg/ml), trypsin (1 mg/ml) and DNAase (0.5 mg/ml).

29. A method as claimed in any one of Claims 23 to 28 characterised in that the aggregate is washed to remove enzymes of the treatment solution.

30. A method as claimed in Claim 29 characterised in that the aggregate is centrifuged and resuspended in physiologically acceptable medium to remove enzymes.

31. A method of treating a patient suffering from neurodegeneration or cerebrovascular disorder characterised in that it comprises implanting an aggregate of hypoxia sensitive cells, these cells releasing neurotransmitters, neuromodulators and/or enzymes, into regions of neurodegeneration or cerebrovascular disorder.

32. A method as claimed in Claim 31 characterised in that the aggregate is as claimed in any one of Claims 1 to 18.

33. A composition characterised in that it comprises an aggregate of hypoxia sensitive cells as claimed in any one of Claims 1 to 18 together with a pharmaceutically acceptable carrier.

34. A composition as claimed in Claim 33 characterised in that it comprises a suspension of from 1 to 100 aggregates as claimed in Claim 1 to 17 in a physiologically acceptable medium.

35. A composition as claimed in Claim 31 characterised in that the medium is a Tyrode medium.

36. A method for the delivery of neurotransmitters, neuromodulators or enzymes, characterised in that it uses an aggregate of hypoxia-sensitive cells that secrete the neurotransmitters, neuromodulators or enzymes as claimed in any one of Claims 1 to 17.

37. A a set of materials and instruments, for preparing aggregates of the invention by the method of the invention, the set preferably being in kit form, comprising

(i) a slicing means dimensioned such that it produces one or more blocks of tissue of from 0.1mm to 0.5mm smallest dimension from a section of donor tissue and

(ii) a composition containing enzymatic components capable of degrading or digesting connective structures in the blocks of donor tissue when in solution form.

38. A set as claimed in Claim 37 characterised in that the slicing means comprises a number of cutting elements that are spaced such as to define tissue block dimensions.

39. A set as claimed in Claim 37 or 38 characterised in that slicing means includes a reception chamber or zone in which the cutting elements are caused to engage the tissue and section it into blocks of the size required for implant.

40. A set as claimed in any of Claims 37 to 39 characterised in that the composition is freeze dried, requiring the addition of fresh liquid, preferably an aqueous liquid such as distilled or de-ionized water, prior to use.

41. A set as claimed in any one of Claims 37 to 40 characterised in that it further comprises one or more of (iii) a vessel for incubating tissue in the enzymatic solution, (iv) a marker device for indicating when the digestion is completed, (v) components suitable in which to suspend the block of enzymatically treated tissue for removal of enzymes and/or for transplant into a host brain and (vi) an implantation device for receipt of the suspended tissue blocks, ie. the aggregates of the invention, and delivery of these said brain.