WO2005016359A1

WO2005016359A1 - Method of promoting remyelination

Info

Publication number: WO2005016359A1
Application number: PCT/CA2004/001502
Authority: WO
Inventors: Michel P. Rathbone; Eva S. Werstiuk; Shucui Jiang
Original assignee: Neurological Technologies Inc.
Priority date: 2003-08-19
Filing date: 2004-08-19
Publication date: 2005-02-24

Abstract

Methods and compositions for promoting the functional recovery and promoting remyelination of the nervous system are provided. The method involves administering an effective amount of a purine nucleoside to an animal in need thereof.

Description

TITLE: METHOD OF PROMOTING REMYELINATION FIELD OF THE INVENTION The present invention relates to methods and compositions for promoting functional recovery and remyelination of the nervous system. BACKGROUND OF THE INVENTION Traumatic spinal cord injury (SCI) induces a prominent local inflammatory reaction and persistent demyelination in the white matter around the lesion site. In humans this demyelination may persist for more than 50 years (1), which slows or disrupts impulse conduction, causing further functional loss. Unfortunately, extensive remyelination does not occur spontaneously. The failure of remyelination is curious, since the spinal cord contains endogenous precursors of the myelin-forming oligodendroglia, which, under certain circumstances, can differentiate into mature oligodendroglia and myelinate axons (2-3). This raises the possibility that after injury there is insufficient signal to trigger the differentiation of the oligodendroglial precursor cells into mature oligodendroglia that are capable of remyelinating axons. Guanosine has a number of trophic effects on a variety of cell types (4- 7). For example, it stimulates proliferation of a variety of cells in culture (4,7) and enhances release of trophic factors, such as NGF, BDNF and bFGF from several cell types (4,6). SUMMARY OF THE INVENTION The present inventors have demonstrated that systemically administered guanosine enhances functional recovery and increases remyelination in the spinal cord of rats with established spinal cord injury. Accordingly, the present invention provides a method of promoting the functional recovery of the nervous system comprising administering an effective amount of a purine nucleoside to an animal in need thereof. The present invention further provides a method of promoting the remyelination of the nervous system comprising administering an effective amount of a purine nucleoside to an animal in need thereof. The present invention also provides pharmaceutical compositions for enhancing the functional recovery and/or promoting remyelination of the nervous system comprising an effective amount of a purine nucleoside in admixture with a suitable diluent and/or carrier. Other features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples while indicating preferred embodiments of the invention are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. BRIEF DESCRIPTION OF THE DRAWINGS The invention will now be described in relation to the drawings in which: Figure 1(a)-(d) are graphs illustrating locomotor functional recovery (a and b), quantification of fast blue stained area at the lesion epicenter of spinal cords (c) and their relation after systemic treatment with guanosine (d). (1a) No further improvement in locomotor performance occurred after 28 days after spinal cord crush. (1 b) Rats that received guanosine (D) showed a marked improvement in locomotor behaviour, which was significantly (p<0.05) better than vehicle-treated control (♦) animals from the third day of treatment onwards. Δ... Δ... , ♦ ... ♦ : Linear regression lines. (1c) The cords from guanosine treated animals had significantly greater myelinated area (*p<0.05; **p<0.01), as determined from the fast blue staining, than did cords from vehicle-treated control animals. (1d) The mean of luxol fast blue stained area at the injury sites plotted against the OWFT scores of individual rats treated with guanosine; the regression line is included. The area of myelination and functional improvement were correlated (r=0.67). Figure 2(a)-(d) are micrographs of cross-sections of spinal cords immunostained for myelin basic protein (MBP) (a and b) and Rip (c and d) from animals treated with guanosine (a and c) or vehicle controls (b and d). Guanosine treatment increased MBP-positive profiles (2a) and Rip-positive mature oligodendrocytes (2c) compared to vehicle-treated control animals (2b and 2d, respectively). DC: dorsal column at the epicenter of the lesion. Scale bar = 60μm for a and b; scale bar = 15μm for c and d. Figures 3(a) and (b) are micrographs of cross-sections of spinal cord segments. Cell proliferation was assessed by incorporation of bromodeoxyuridine (BrdU) into DNA. BrdU immunostaining on sections of spinal cords from guanosine- (a) and vehicle- (b) treated animals following chronic spinal cord crush at the lesion site shows that the number of BrdU immunolabeled nuclei, and hence the number of mitotically active cells, was higher in cords of guanosine treated rats. This was particularly apparent in the dorsal columns at the epicentre of the crush site. IC: injury center; DC: dorsal column. Scale bar=45μm for both (a) and (b). Figures 4(a) and (b) are micrographs of cross-sections of spinal cord segments immunostained for NG2. The sections of spinal cords from guanosine-treated animals (a) had NG2 immunopositive cells throughout the lesion site; but particularly in the dorsal columns of the white matter. The NG2 immunopositive cells exhibited unipolar, bipolar and mutlipolar morphologies. Compared to cords from guanosine-treated animals, those from control animals (b) had far fewer NG2 immunopositive cells. IC: injury center; DC: dorsal column; GM: grey matter. Scale bar = 45μM for both (a) and (b). Figure 5 (left and right panels) are confocal fluorescent micrographs using antibodies against BrdU (green) and NG2 (red). The left panel shows double staining for the vehicle-treated control cord and the right panel shows double staining for the guanosine-treated cord. Sections of spinal cords from guanosine-treated animals (right) had BrdU (+) (green) and NG2 (+) (red) double-labeled cells throughout the lesion site. Compared to cords from guanosine-treated animals, those from control animals (left) had significantly fewer labeled cells. Scale bar = 50μM for both left and right panels. Figure 6(a) and (b) are mircographs of cross-sections of spinal cord segments immunostained for OX-42. Macrophages and microglia are OX-42 positive. OX-42 positive cells were found throughout white matter around the lesion site in cords from both guanosine (a) and vehicle treated (b) animals. There was no difference in the number of OX-42-immnuoreactive cells between sections obtained from guanosine-treated and vehicle-treated control animals. DC: dorsal column. Scale bar = 50μM for both (a) and (b). DETAILED DESCRIPTION OF THE INVENTION The present inventors have demonstrated that systemically administered guanosine enhances functional recovery and increases remyelination in the spinal cord of rats with established spinal cord injury. Accordingly, the present invention provides a method of promoting the functional recovery of the nervous system comprising administering an effective amount of a purine nucleoside to an animal in need thereof. The present invention also provides a use of an effective amount of a purine nucleoside to promote the functional recovery of the nervous system. The invention further provides a use of an effective amount of a purine nucleoside in the manufacture of a medicament to promote the functional recovery of the nervous system. The present invention further provides a method of promoting the remyelination of the nervous system comprising administering an effective amount of a purine nucleoside to an animal in need thereof. The present invention further provides a use of an effective amount of a purine nucleoside to promote remyelination of the nervous system. The present invention further provides a use of an effective amount of a purine nucleoside in the manufacture of the medicament to promote remyelination of the nervous system. The term "effective amount" as used herein means an amount effective, at dosages and for periods of time necessary to achieve the desired result, e.g. promoting functional recovery and/or promoting remyelination. The term "animal" as used herein includes all members of the animal kingdom, including humans. Preferably, the animal to be treated is a human. The term "the nervous system" includes both the peripheral and central nervous systems. The methods of the invention to promote the functional recovery and/or promote the remyelination in the nervous system can be used to treat any disease or condition of the central nervous system and/or peripheral nervous system wherein it is desirable to regain function and/or remyelination of the nervous system. Such diseases or conditions include, but are not limited to, the demyelinating diseases such as multiple sclerosis and other neural diseases in which demyelination exerts a prominent source of dysfunction, including, but not limited to, neuromyelitis optica, acute disseminated encephalomyelitis, acute and subacute necrotizing hemorrhagic encephalitis and subacute necrotic myelopathy, stroke, trauma to the nervous system, anoxia induced through any means, damage induced by radiation, including therapeutic radiation, and demyelinating disorders of the peripheral nerves, including Bell's Palsy, compression injuries to nerves, and peripheral polyneuropathies such as acute and chronic demyelinating polyneuropathies and demyelination of nerves associated with any other disorder, including mononeuritis multiplex. Accordingly, the present invention provides a method of treating a disease or condition wherein it is desirable to promote functional recovery or remyelination of the nervous system comprising administering an effective amount of a purine nucleoside to an animal in need thereof. The present invention further provides a use of an effective amount of a purine nucleoside to treat a disease or condition wherein it is desirable to promote functional recovery of the remyelination of the nervous system. The present invention also provides a use of an effective amount of a purine nucleoside in the manufacture of a medicament to treat a disease or condition wherein it is desirable to promote functional recovery of remyelination of the nervous system. In a preferred embodiment, the method is used to treat a spinal cord injury. The term "treatment or treating" as used herein means an approach for obtaining beneficial or desired results, including clinical results. Beneficial or desired clinical results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, diminishment of extent of disease, stabilized (i.e. not worsening) state of disease, preventing spread of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. "Treating" can also mean prolonging survival as compared to expected survival if not receiving treatment. The purine nucleosides used in the methods of the invention can be any purine nucleoside such as guanosine, inosine, adenosine and analogs thereof. Examples of analogs are provided below. Derivatives by modifying the 2-amino-NH₂ group of guanosine: 2 - methyl - 2-CH₃- 2 -ethyl- 2-C₂H₅- 2 - ethylamino - 2- CH₃CH₂NH - N,N- dimethyl- 2-(CH₃)₂N- 2 - methylamino - 2 - CH₃NH - 2 - ethylamino - 2 - CH₃CH₂NH - N₂ - benzoyl - 2 - C₆H₅C - NH - phenyl - amino - 2 - CβHsNH - Substituting - phenyl - amino - Derivatives by modifying the 6 - keto - C=0 group of guanosine or inosine: 6 - thio - 6 - SH - 6 -amino- 6-NH₂- 6 - chloro - 6 - CI - 6-methoxy- 6-OCH3- 6 - cyclopentyl - 6 - C₅H₉ - 6 - cyclohexyl - 6-C₆Hn- Derivatives by substituting the imidazole ring of the purines: N7 - methyl - 7 - CH₃ - C8 - hydroxy - 8 - OH - C8 - bromo - 8 - Br - Derivatives by modifying the ribose ring of purine nucleosides: 2' - deoxy - ribose 2', 3' - dideoxy - ribose 5' - carbamido - derivatives Changing the ribose to other sugars - eg: arabinose Changing the ribose to carbocyclic analogues - eg: cyclopentane The pharmaceutical compositions can be prepared by per se known methods for the preparation of pharmaceutically acceptable compositions which can be administered to patients, and such that an effective quantity of the active substance is combined in a mixture with a pharmaceutically acceptable vehicle. Suitable vehicles are described, for example, in Remington: The Science and Practice of Pharmacy (20^th Edition, ed. A.R. Green, Lippincott Williams & Wilkins, Baltimore, MD, USA, 2000). On this basis, the pharmaceutical compositions include, albeit not exclusively, the active compound or substance in association with one or more pharmaceutically acceptable vehicles or diluents, and contained in buffered solutions with a suitable pH and iso-osmotic with the physiological fluids. The pharmaceutical compositions may additionally contain other agents such as other agents that can prevent the inhibition of apoptosis or that are used in treating inflammatory conditions or sepsis. Such pharmaceutical compositions can be for intralesional, intravenous, topical, rectal, parenteral, local, inhalant or subcutaneous, intradermal, intramuscular, intrathecal, transperitoneal, oral, and intracerebral use. The composition can be in liquid, solid or semisolid form, for example pills, tablets, creams, gelatin capsules, capsules, suppositories, soft gelatin capsules, gels, membranes, tubelets, solutions or suspensions. The pharmaceutical compositions of the invention can be intended for administration to humans or animals. Dosages to be administered depend on individual needs, on the desired effect and on the chosen route of administration. The present invention also provides pharmaceutical compositions for enhancing the functional recovery and/or promoting remyelination of the nervous system comprising an effective amount of a purine nucleoside in admixture with a suitable diluent and/or carrier. The following non-limiting example is illustrative of the present invention: EXAMPLES

Example 1 : Guanosine Enhances Remyelination MATERIALS AND METHODS All experiments were performed in compliance with the requirements of the Animals for Research Act of Ontario, Canada and the guidelines of the Canadian Council on Animal Care (CCAC), and had been approved by the Animal Research Ethics Board (AREB) of McMaster University. Surgical procedures. Adult female Wistar rats (280-300g weight, Charles River) were anaesthetised with isoflurane (3-5%): 0₂ (1L/min). Buprenorphine (0.03 mg/kg body weight, subcutaneously) was administered prior to surgery for pain relief. Spinal cords were surgically exposed and crushed with modified coverslip forceps (9-10) producing a moderate spinal cord injury (10). Briefly, the backs of anaesthetized animals were shaved and cleaned with proviodine and alcohol. The vertebrae were exposed and a dorsal laminectomy performed at T12 to expose the cord, leaving the dura intact. The forceps were closed slowly (over 2 seconds) over the entire width of the spinal cord, to a thickness of 1.4 mm, held closed for 15 seconds and then removed. Wounds were closed by suturing the vertebral muscles and fat pad over the wound and closing the skin with wound clips. Postoperatively, rats were kept quiet and warm. Tylenol was given if required. Behavioral assessment and drug administration. After crushing the spinal cord, locomotor recovery was assessed weekly for 5 weeks with an open field walking task (OFWT), scored using a standard scale (11), on which 0 = no movement at all and 21 = normal function. After 4 weeks no further spontaneous recovery occurred (Figure 1). In the inventors' experience, about 90% of rats (n=24) with this degree of crush recover and stabilize within the range of 9.7 to 11.5 on the locomotor function scale. On day 35 after crush injury, 24 rats performing in this range were randomly assigned to two groups. One group (n=12) received daily intraperitoneal injections (ip) of guanosine. Eight mg of guanosine (Sigma-Aldrich, Louis, MO) was dissolved in 10 μl of 0.1N NaOH, then added to 990 μl saline to make an 8 mg/ml solution. Guanosine was given ip (8 mg/kg body weight) from day 35 through 41. The other group (n=12) received equivalent volumes of saline by i.p. injection. Only the investigator who prepared the drug solutions was aware of the treatment. That individual was blinded to all other parts of the experiment. Other investigators remained blinded to treatment groups until data analysis was completed. Locomotor function of each rat was tested daily, immediately prior to the first treatment and at 3 and 24 hours after each treatment. Histochemistry. On post-operative day 42, immediately following the final locomotor testing, rats were deeply anaesthetised and perfused transcardially with 4% paraformaldehyde (PFA) in phosphate buffered saline (PBS, pH 7.4). Segments of the spinal cords from T9 to L1 were removed and cryo-protected in 30% sucrose/PBS solution. A segment of each cord, extending from 5 mm rostral to 5 mm caudal to the lesion site was embedded in medium (Tissue- Tek® O.C.T. compound, Sakura Finetek U.S.A., Inc., Torrrance, CA). Serial transverse sections were cut at 15-20 μm intervals on a cryostat and mounted onto slides (ColorFrost/Plus; Fisher, Pittsburgh, PA). Every fifth section was stained with Luxol fast blue, a lipophilic dye commonly used to stain myelin (12). Adjacent sections were stained immunohistochemically after thorough rinsing with PBS and several steps to reduce background staining (13-14). Mature oligodendrocytes in the injured spinal cords were identified with Rip, a specific marker for mature oligodendrocytes which recognises an unknown epitope (15) and which has been used successfully in the CNS to label both oligodendrocyte processes and myelin sheaths (13,15,16). Immunostaining for myelin basic protein (MBP) (13,16) or Rip was performed as described by other investigators (13,15). Briefly, sections were incubated with primary polyclonal antibodies to MBP (1:50; Chemicon Int., Temecula, CA) or monoclonal Rip antibodies (1 :200; Chemicon Int., Temecula, CA), at 4°C overnight. Following washing, sections were incubated with biotinylated goat anti-rabbit IgG (1 :400) for polyclonal MBP antibody or biotinylated horse anti-mouse IgG (1 :400) for monoclonal Rip antibody, respectively. Sections were then incubated with Elite avidin-biotinylated- complex (ABC; Vector Laboratories, Burlingame, CA) for 1 hr, followed by 3,3'-diaminobenzidine (DAB) substrate (Vector Laboratories, Burlington, ON, Canada) for 5-10 min. Sections were dehydrated, coverslipped, and examined under a microscope. Quantification. To quantify the luxol fast blue stained portion, the luxol fast blue stained section from each cord (n=10 for each group) that contained the greatest lesion area was selected together with the adjacent 2 caudal and 2 rostral luxol blue stained sections. Six additional unoperated normal animals were used to assess the magnitude of recovery. Digital photographs were taken of the sections. The total area of each section and the area of the fast blue-stained portion were measured using a computerised Bioquant BQ- TCW98 image analysis program by an investigator who was blind to group assignment. For quantification of Rip-positive cells, 5-7 sections from each animal (n=10 for each group) at the lesion site (every third section, 100 μm apart) were analysed. Five additional unoperated normal animals were used to assess the magnitude of recovery. The number of Rip-labelled cells was determined by manual counting. Data are expressed as the number of immunostained cells per section (mean±SEM). Statistical analysis. Statistical analyses were performed on a Macintosh computer using GB-Stat ppc 6.5.2. Behavioural scores and histologic quantification were analysed by the Kruskal-Wallis non-parametric analysis of variance (ANOVA). Post hoc comparisons were made using the Dunnett's test. Correlation between behavioral and histological outcomes was analysed using regression analysis. RESULTS

Guanosine treatment enhanced locomotor functional recovery. After a spinal cord crush of moderate severity motor function of the legs of rats recovered progressively, albeit incompletely, for 3 - 4 weeks, after which recovery plateaued, and no further improvement was observed (8). This plateau persisted in control rats that received vehicle injections from day 35 to day 42 after the crush (Figure 1b). In contrast, rats treated daily with intraperitoneal injections of guanosine beginning 35 days after injury, showed a progressive improvement in locomotor performance over the next week, from a mean score on the locomotor scale of 10.8 to 11.7 (Figure 1b). The locomotor function of the guanosine-treated animals was statistically better than that of controls from the third day of treatment onwards (p<0.05). Over the course of the 7-day treatment the saline treated rats remained at the stage of having only occasional weight-supported plantar steps, with no fore limb- hind limb coordination. In contrast, the guanosine treated rats had consistent weight-supported plantar steps with occasional to frequent fore limb-hind limb coordination. Guanosine treatment increased myelination. The progressive nature of the improvement in motor function in the guanosine-treated rats indicated that guanosine was having cellular effects on the spinal cord rather than pharmacologically enhancing neurotransmission. Histological examination of the rat spinal cords confirmed this. In rats treated with guanosine for seven days, the area stained by fast blue (presumptive myelin) at the lesion epicentre was significantly greater (p<0.01) than in vehicle-treated animals (Figure 1c). The luxol fast blue stained area in the injured cords from animals treated with guanosine correlated with their behavioural recovery in the open field walking scores, correlation coefficient of 0.67 (r=0.67), indicating that the recovery of function may be related to the increase in myelin (Figure 1d). To confirm that the fast blue stained areas were myelin, the inventors immunostained sections with anti-myelin basic protein (MBP), an integral membrane protein in myelin. The spinal cords of guanosine-treated animals contained substantially more MBP-labelled profiles at the injury site than control animals (Figures 2a and b).

Guanosine treatment increased the number of Rip-positive cells. T o determine whether the increased myelin in the cords of rats treated with guanosine was associated with mature oligodendrocytes, the inventors identified mature oligodendrocytes with a monoclonal antibody to Rip, a specific marker for nascent and mature oligodendrocytes (13,15,16). Rip intensely immunostained oligodendrocyte cell bodies and their processes (Figure 2c and d). Spinal cords of rats that had been treated with guanosine for 7 days had significantly more Rip-positive cells (452±36/section) in the epicentre compared to spinal cords from animals that received only vehicle (208±25/section; p<0.01). Corresponding sections from 5 unoperated normal spinal cords contained 1005±75 oligodendrocytes/section. DISCUSSION In the present study, 5 weeks after traumatic injury of the spinal cord of rats, when recovery of function had plateaued, systemic administration of guanosine enhanced functional recovery. This improved function correlated with increased remyelination. Myelin is essential for normal nerve impulse conduction so that loss of the myelin sheath around axons may significantly contribute to the neurological deficits after SCI. Consequently, functional recovery may depend, at least in part, on remyelination (18). These preliminary data indicate a possible causal relationship between remyelination induced by guanosine and the concomitant improvement in locomotor function. The first unequivocal evidence of spontaneous myelin repair in the mammalian CNS was reported by Bunge et al. (17) and confirmed by others (18). However, remyelination is characteristically incomplete after injury to the CNS (18-19). Oligodendrocyte precursors proliferate in the first two weeks after acute spinal cord injury in rats, but remain "silent" thereafter (14,20). However, those endogenous progenitors are able to differentiate into mature cells capable of myelinating axons (2-3). Attempts to promote myelin repair have, therefore, focused on stimulating or enhancing this natural process. Successful manipulation of the glial response to injury, enabling stable restoration of structure and function, is a fundamental goal in the treatment of CNS damage. The administration of neurotrophic factors provides one method of manipulating glial response. Several studies in vitro have demonstrated that various trophic factors can a) stimulate proliferation of oligodendrocyte progenitors (21 -22); b) dedifferentiate mature oligodendrocytes (23-24); c) cause myelination in vitro (25). McTigue et al. (13) showed that in vivo administration of growth factors, neurotrophin-3 (NT-3) and brain-derived neurotrophic factor (BDNF), via genetically engineered fibroblasts promoted oligodendrocyte proliferation and axonal sprouting. The mechanisms by which guanosine facilitates remyelination after chronic traumatically-induced SCI are unknown. However, as guanosine stimulates several cell types to produce and release trophic factors, including bFGF, NGF and NT-3 in vitro as well as in vivo following traumatic injury (4), the effects of guanosine on remyelination and on recovery of function may be mediated by one or more of these growth factors. The data show that systemic treatment with guanosine increased the number of mature Rip-positive oligodendrocytes in the region of the lesion, consistent with increased remyelination. This indicates that guanosine may induce remyelination by stimulating "silent" endogenous oligodendrocyte progenitors to proliferate and to differentiate into mature oligodendrocytes. Alternatively, the increased number of oligodendrocytes may result from stimulation of their migration from surrounding regions. The inventors are investigating these possibilities. CONCLUSION These studies demonstrate a novel finding. In the spinal cords of rats with established, stable, spinal cord injury systemically administered guanosine enhances function with concomitant increase in the number of mature oligodendrocytes and myelination. The possibility exists that guanosine may have similar beneficial effects in higher animals and humans with stable, incomplete paraplegia resulting from spinal injuries.

Example 2: Guanosine Increases the Number of NG+ Cells NG2 is a cell surface antigen, chondronitin sulphate proteoglycan, and has been used to identify oligodendrocyte progenitor cells in adult mammalian CNS (review: Dawson et al., 2000). NG2-positive (NG2+) cells can be detected after CNS injury and play a role in remyelination (Carroll et al., 1998; Keirstead et al., 1998; Levine and Reynolds, 1999; McTigue et al., 2001; review: Dawson et al., 2000). In most cases NG2+ cells are likely oligodendroglial precursors (Levine and Stallcup, 1987; Keirstead et al. 1998; McTigue et al. 2001), but may include other cell types: macrophages, microglia and infiltrating Schwann cells (McTigue et al. 2001), precursors of astrocytes (Levine and Stallcup, 1987). The inventors have found that cell proliferation (assessed by incorporation of bromodeoxyuridine, BrdU, into DNA) was higher in the cords of rats treated with guanosine (Fig. 3). In addition, the inventors have found that the number of NG2-reactive cells was also higher (Fig. 4) after guanosine treatment. Guanosine enhanced NG2 cell proliferation was shown using double fluorescent staining (Fig. 5). The inventors discovered that immunostaining by OX-42, a marker for microglia and macrophages, did not increase following guanosine treatment (Fig. 6), raising the possibility that the NG2+ cells might be oligodendrocyte progenitor cells.

Guanosine treatment stimulated cell division in demyelinated areas of spinal cords with chronic stable injury Cellular proliferation was examined by administering the thymidine analogue, bromodeoxyuridine (BrdU; 50 mg/kg, i.p. Sigma, B-5002), daily from day 35 to 41 after to the crush. On postoperative day 42, immediately following the final locomotor testing, the rats were anaesthetized with sodium barbital (75-mg/kg-body weight), perfused transcardially with 4% paraformaldehyde and a segment, T9 to L1 , was removed from each spinal cord. The tissues were processed histologically (McTigue et al., 2001). A segment of each cord including the lesion site plus 10 mm rostral and caudal to the lesion site was embedded in Tissue Tek^® medium. Serial sections were cut at 15-20 μm intervals on a cryostat and mounted onto slides (ColorFrost/Plus; Fisher, Pittsburgh, PA). BrdU immunohistochemistry was performed as described by McTigue et al. (2001; 2002). Briefly, sections were rinsed thoroughly with phosphate buffered saline (PBS). After several steps to reduce background staining (McTigue et al., 2001), sections were incubated with primary antibody (mouse monoclonal anti-BrdU; 1:200; NCL-BrdU; Novocastra laboratories Ltd, UK) at 4°C overnight. Following washing, sections were incubated with biotinylated horse anti mouse IgG secondary antibodies. Sections were then incubated with Elite ABC (Vector Laboratories, Burlingame, CA) for 1 hr, followed by DAB substrate kit for peroxidase (Vector Laboratories) for 5-10 min. Sections were dehydrated and coverslipped and examined under a microscope. Figure 3 shows that the number of BrdU immunolabeled nuclei, and hence the number of mitotically active cells, was higher in cords of guanosine treated rats. This was particularly apparent in the dorsal colums at the epicenter of the crush site. IC: injury center; DC: dorsal column. Scale bar=45μm for both (a) and

(b). Guanosine treatment for 7 consecutive days increased the number of NG2 immunopositive cells in demyelinated areas of spinal cords with chronic stable injury The immunohistochemical procedures in these experiments were similar to those described in detail in Figure 3 above, except that the first antibodies were polyclonal NG2 antibodies (1 :200; Chemicon Int. Temecula) and the second antibodies were biotinylated goat anti-rabbit (1:400). Figure 4 shows that sections of spinal cords from guanosine-treated animals (a) had NG2 immunopositive cells throughout the lesion site; but particularly in the dorsal columns of the white matter. The NG2 immunopositive cells exhibited unipolar, bipolar and mutlipolar morphologies. Compared to cords from guanosine-treated animals, those from control animals (b) had far fewer NG2 immunopositive cells. IC: injury center; DC: dorsal column; GM: grey matter. Scale bar = 45μM for both (a) and (b). Guanosine treatment stimulated NG2-positϊve progenitor proliferation in demyelinated areas of spinal cords with chronic stable injury The immunohistochemical procedures in these experiments were similar to those described in detail in Figure 3 above, except that the first antibodies were mouse monoclonal anti-BrdU or polyclonal NG2 antibodies and the secondary antibodies were either FITC-conjugated donkey anti- mouse (green) (1 :200; Chemicon Int. Temecula) or RITC-conjugated donkey anti-rabbit (1 :200; Chemicon Int. Temecula). To determine the proliferation of progenitors, the inventors used double fluorescent staining with antibodies against BrdU and NG2. Sections of spinal cords from guanosine-treated animals (right) had BrdU (+) (green) and NG2 (+) (red) double-labeled cells throughout the lesion site. Compared to cords from guanosine-treated animals, those from control animals (left) had significantly fewer labeled cells. Scale bar = 50μM for both (a) and (b). OX-42 expression Immunohistochemical techniques were similar to those described in Figure 3, except that the first antibody was a monoclonal OX-42 antibody (1 :200; CD11b, Research Diagnostics INC. Flanders NJ. OX-42). To determine whether guanosine treatment increased the number of macrophages and microglia, the number of OX-42 positive cells in guanosine treated and control sections was observed. Macrophages and microglia are OX-42 positive. OX-42 positive cells were found throughout white matter around the lesion site in cords from both guanosine (a) and vehicle treated (b) animals. There was no difference in the number of OX-42-immnuoreactive cells between sections obtained from guanosine-treated and vehicle-treated control animals. DC: dorsal column. Scale bar = 50μM for both (a) and (b). While the present invention has been described with reference to what are presently considered to be the preferred examples, it is to be understood that the invention is not limited to the disclosed examples. To the contrary, the invention is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims. All publications, patents and patent applications are herein ncorporated by reference in their entirety to the same extent as if each ndividual publication, patent or patent application was specifically and ndividually indicated to be incorporated by reference in its entirety. FULL CITATIONS FOR REFERENCES REFERRED TO IN THE SPECIFICATION

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Claims

WE CLAIM:

1. A use of an effective amount of a purine nucleoside in the manufacture of a medicament to promote functional recovery of the nervous system.

2. A use of an effective amount of a purine nucleoside in the manufacture of a medicament to promote remyelination of the nervous system.

3. A use of an effective amount of a purine nucleoside in the manufacture of a medicament to treat a disease or condition wherein it is desirable to promote functional recovery or remyelination of the nervous system.

4. A use according to any one of claims 1 to 3 wherein the purine nucleoside is selected from guanosine, inosine,_. adenosine or an analog thereof.

5. A use according to claim 4 wherein the purine nucleoside is guanαsine or an analog thereof.

6. A use according to any one of claims 1 to 5 for use in treating a spinal cord injury.