WO2005038453A1 - Early assessment of motor recovery in spinal cord injury patients - Google Patents

Abstract

Prognosis of an acute neuropathy (myelopathy) can be made by assessing the levels of markers taken from body fluid or tissues samples of patients at an early stage from diagnosis. This is applicable in particular to spinal cord injury (SCI) where bone resorption markers are demonstrated to provide bimodality. The bimodality is particularly apparent where ratios of bone resorption markers measured in early stage injury can be aligned with later conducted ASIA score AB and CD loss of function assessments.

Description

EARLY ASSESSMENT OF MOTOR RECOVERY IN SPINAL CORD INJURY PATIENTS

This invention relates to early assessment of the prognosis of motor recovery for victims of spinal cord injury (SCI) or other central nervous system (CNS) injuries or pathologies.

BACKGROUND OF THE INVENTION

Spinal cord injury (SCI) can result from a number of causes that may be either traumatic or medical. Traumatic neuropathology may exhibit as contusion, compression or transection and often is due to vertebral fracture(s) and or dislocation(s), but can occur in the absence of fracture. Medical injury may be neoplastic, vascular, infective, or due to malformation within the spinal canal (spinal stenosis) or, malformation within the cord per se (syrinx). SCI may be caused by exposure to neurotoxins, or electrical or hypobaric injury or, occur as a complication of a critical illness or a surgical procedure, however, the lesion must occur within the neural canal. For example, SCI does not include bracbial plexus lesion or peripheral nerve injury outside the neural canal.

The SCI may result in a an epicentre of primary ischaemic tissue damage and (delayed) appearance of a penumbra with features of ischemic and apoptotic cell death, which is a typical early histopathological finding in most traumatic myelopathies. After traumatic SCI apoptotic cells and patches of axon demyelination also can be seen remote from the injury site. Multifocal lesions or diffuse tissue damage occurs where the spinal lesion results, for example, from neurotoxic, electrical or hypobaric exposure, high impact trauma, or a neurological disorder, for example, auto-immune disorders (multiple sclerosis [MS]), other neurodegenerative conditions (amyotrophic lateral sclerosis [ALS]) or infective disorders (transverse myelitis). However, there is considerable overlap between secondary injury mechanisms after traumatic myelopathy and the pathophysiology of the common neuro-inflammatory disorders (MS, ALS, and Alzheimer's disease [AD]). Apoptosis of neurons and oligodendrocytes (the oligodendrocyte is primarily responsible for axon myelination in the longitudinally oriented spinal long tracts) results in secondary demyelination, with attendant inflammatory responses. Hence, in traumatic cases, histopathological findings can include diffuse sites of secondary demyelination frequently at sites remote from the area of primary tissue damage perhaps as a result of oligodendrocyte susceptibility to proapoptotic mediators with attendant microglial activation.

Downstream of injury, the secondary cellular inflammatory responses to injury involves accumulation of resident cells (microglia, reactive astrocytes) and infiltrating immune cells (neutrophils, monocytes, and lymphocytes reactive for myelin products). Thus after CNS trauma, intra-axonal pathology can include cyto-skeletal and axo-lemmal abnormalities, local axonal failure, impaired axonal transport, axonal swelling and detachment, and in addition, secondary auto-destructive processes, involving demyelination of intact axons located below the lesion. It is well recognised that the damaged human central nervous system CNS has limited regenerative potential after critical periods of development. The limited potential for restoration of function is in part a result of the resident cells (microglia, reactive astrocytes) presenting both a chemical inhibitory and physical mechanical barrier to path-finding by sprouting fibres, which inhibits the formation of functional connections (axon regeneration). An additional reason is because the human brain and spinal cord have limited potential for spontaneous cell replacement neurogenesis) and therefore the subsequent physiological repair after tissue damage is non-functional.

The progression of SCI pathophysiology following onset can be categorised into a number of sequential phases.

The initial pathophysiology may additionally be considered in two parts: primary injury (microvascular injury, ischaemia, vasogenic oedema, cell oedema and necrosis) and secondary injury (an initial period of about 21 days when secondary auto-destruction of neural tissue occurs with attendant metabolic cascades). (The temporally regulated phases of secondary damage consist of: glutamate excitoxicity, , cell apoptosis (pi 5 or fas receptor activation of pro-apoptotic proteins, (calpain and caspase cysteine protease activation) with attendant loss of neurons and oligodendrocytes, and activation of the cell inflammatory response (by phagocytotic cells- resident microglia and immune cells). During secondary pathophysiology, the course of disruption to endothelial- spinal cord barrier (E-SC) permeability, is an important determinate of the composition the internal environment of the spinal cord. Studies of E-SC barrier diffusion restraints and efflux/influx mechanisms, indicate that an initial period of disturbed E-SC permeability facilitates the exchange of immune cells, macromolecules and mediators between the spinal cord and systemic circulation. The exchange of ions, pro-inflammatory mediators, metabolites and neurotransmitters has significant implications for diffusion within the extracellular space (ECS); and neuron- glial communication.

The histopathology includes gliotic scar formation, which enlocates the central haemolytic cyst. The gliotic scar presents a physical and chemical barrier (via growth inhibitory molecules- eg, myelin associated glycoproteins [MAG, NOGO], chrondroitan sulphate proteoglycans [CSPGs]) and is implicated in growth cone collapse. The inhibitory gliotic scar forms over a period of weeks. The period of cell inflammatory response and the question of whether reactive astrocytes and infiltrating immune cells are neuro-protective or exacerbate neuro-degeneration, is relatively controversial. Models of autoimmune disease (eg acute autoimmune encephalitis [AEA]), suggest that the T- cell mediates secondary axon demyelination, and in this way have indicated potential for an autoimmune response after injury. Demyelination entails an inflammatory process, including trafficking of immune cells (neutrophils, monocytes and T- lymphocytes reactive with a variety of myelin antigens) into the spinal cord and pro- inflammatory cytokine activation.

It is well known that elevated pro-inflammatory cytokines [interleukin- 1, 6, 11 (II- 1,6,11) and tumor necrosis factor - alpha (TNF- alpha)] in cerebro-spinal fluid (CSF) levels are hallmark signs of CNS injury. These cytokines have been implicated in the processes of neuro-destruction (demyelination, axonal injury, apoptosis) and neuro- restoration (neural repair, re-myelination and revascularisation) in the CNS micro- environment. After CNS injury, these cytokines are actively transported to the systemic circulation. Experimental data also indicate that elevated CSF levels of pro-apototic mediators induce bone loss. In the bone marrow micro-environment, pro-inflammatory cytokines are responsible, for the differentiation and activation of osteoclasts. Receptor activated nuclear transcription factor kappa- B ligand [RANK/RANKL] is an essential cytokine for ostoeclastogenisis. Osteoprotegerin (OPG), a decoy receptor for RANKL, physiologically counterbalances osteoclast differentiation and activation, and is a key molecule in the regulation of the balance of bone resorption and formation. OPG, a member of the TNF-alpha receptor superfamily, also has a high affinity for pro- inflammatory cytokines in the systemic circulation, and T-cell activation (for example, in the pathogenesis of rhematoid arthritis, multiple myeloma) enhances the serum ratio of receptor-activated nuclear transcription factor kappa- B ligand (RANKL) to osteoprotegerin (OPG). Glutamate signalling also has been identified in osteoblasts and onteoclasts, although the physiological role(s) of glutamate machinery in bone have not been elucidated. It is postulated that, like other markers of bone resorption identified in the experiments (see accompanying figures) the serum ratio of RANKL:OPG will be disturbed initially after SCI in the period corresponding to immune cell activation and disruption to E-SC barrier permeability. In the interpretation, the time to peak amplitude of bone resorption after SCI corresponds to the underlying CNS pathophysiology, and the magnitude of peak amplitude, to the severity/extent of spinal cord tissue damage.

Secondary axon degeneration contributes significantly to the extent of spinal cord tissue damage, and is highly correlated to degree of impairment (and pattern of functional recovery). Experimental data have indicated that secondary demyelination takes place over a period of days to weeks to typically peaking at about 3 weeks from injury. However clinical data suggest that demyelination after SCI may progress over a period of months to several years. Significantly, demyelination affects otherwise intact axons below the injury and therefore greatly contributes to functional impairment after SCI.

The clinical course of SCI and its early treatment is well described. The cardinal signs include: hypotonus, hypotension, hypoxia and hypothermia. An initial phase of spinal shock or "diaschasis" is characterised by H-reflex depression (of variable in duration). A period of reflex recovery (but not necessarily functional recovery) follows the initial period of spinal shock. The degree of functional recovery depends greatly on the severity of tissue damage (in the epicentre and penumbra), the extent of secondary demyelination (of intact axons below the lesion) and, the inherent potential for neuroplasticity inbuilt into the central nervous system (CNS), to circumvent the injury, a mechanism which is known to be more robust earlier in life. The mechanisms of functional recovery may include: sprouting of remaining fibres; a rewiring, formation of de novo connections via new synapses which may or may not be aberrant in their strength, direction and new neurochemistry. There is a belief that environmental enrichment (EE) may either strengthen plasticity throughout the CNS or, trigger compensatory mechanisms, which is a general principle of therapy Re-organisation of undamaged tissue in higher brain centres also is believed to occur, and, bone marrow fMRI evidence suggests that cortical motor and/or sensory processes are re-organised to accommodate a "de novo" paradigm of functional organisation.

Following the finite period for recovery, there is a phase of chronic impairment, where the victim's neurological status may be relatively stable. The net resultant remaining injury (as defined by functional impairment) is typically measured by a functional test in terms of the locus of injury (segmental level) and then classified by diagnosis (whether the victim has tetraplegia (Cl-Tl segment) or paraplegia (T2 segment and below). The extent of the injury (whether it is clinically complete or incomplete), is an important determinant of clinical outcome. The overall health of the individual such as whether there are co-morbidities, associated injuries (eg; brain injury, extra- vertebral fracture) or secondary complications, may all independently contribute to the final clinical picture. Typically the recovery period is believed to end at about 2 years from SCI diagnosis, although late functional recovery sometimes may occur after that time.

Broadly there is an inverse association between the segmental level and the extent of functional impairment. Clinically, segmental level is evaluated by a system of classification, in which: "the segmental level at which the spinal sensory nerves and axons leave the spinal cord, via segmental nerves and roots is determined. The roots are numbered and named according to the foramina though which they enter/exit the vertebral column. For example the two C6 roots (left and right) pass though the foramina situated between the C5 and C6 vertebrae" p3 International Standards for Neurological and Functional Classification of Spinal Cord Injury, (1996). A lesion in the cervical or high thoracic region (Cl-Tl) leads to "tetraplegia" (also known as quadriplegia). By international definition the term tetraplegia refers to impairment or loss of function in the cervical or first thoracic segment of the spinal cord, due to damage of neural elements in the spinal canal.

Complete injury at C2 or above paralyses the muscles of ventilation, all four limbs and trunk, and pelvic organs.

Complete injury at C4 to Tl paralyses the muscles of the trunk, all four limbs, and pelvic organs.

Lesion in the thoracic region at T2 and below leads to paraplegia. By international definition the term paraplegia refers to impairment or loss of function in the thoracic, lumbar or sacral (but not cervical) segments of the spinal cord, due to damage of neural elements in the spinal canal.

Complete injury at T2 to T12 paralyses the muscles of the trunk (for levels at or above T6), and for the remainder, of both lower limbs, and pelvic organs.

Lesion in the lumbar region (below T12) leads to paraplegia and depending on the , segmental level, patients may have some motor and sensory sparing in the lower limbs, but not necessarily of pelvic organs (S 4-5).

Lesions at or below T12 are not strictly an injury of the spinal cord per se but, are lesions of the sacral and lumbar nerve roots in the spinal canal (the conus medullaris or cauda equina ).

Patients with lesions at or below the T12 level frequently exhibit flaccid bladder, bowel and lower limbs, and if sacral, may have preserved reflexes in pelvic organs (of micturition and bulbo-cavernous reflex). However these lesions are contemplated as SCI for purposes of the present invention. Thus there is a high degree of heterogeneity in the (neuro)pathology and attendant functional variability in any population of SCI sufferers.

The degree of functional disability for each of the above can be further classified either as clinically complete, discomplete (an electrophysiological definition) or incomplete. The extent of tissue injury most frequently is classified by physical and/or electrophysiological evaluation (see explanations below).

At present clinical SCI is classified by physical examination according to the American Spinal Injury Association. International standards for neurological and functional classification of spinal cord injury,. Chicago: Association; (first edition Stover et al 1992, revised 2000). This is known as the American Spinal Injuries Association (ASIA) score (Stover et al 1996, Ditunno et al 2000) where individuals are ranked by assessment of dermatome (the area of skin innervated by the sensory axons within each segmental nerve root) and myotome (the collection of muscle fibres innervated by axons within each segmental nerve root) as follows: A - Complete: No motor or sensory function is preserved in the lowest sacral segment (S5) (ie, no sacral reflexes) B - Incomplete: Sensory but not motor function is preserved below the neurological level and includes the sacral segments (S4-5) C - Incomplete: Motor function is preserved below the neurological level, and more than half of key muscles below the neurological level have a muscle grade less than 3 D - Incomplete: Motor function is preserved below the neurological level, and at least half the key muscles below the neurological level have a muscle grade of 3 or more E - Normal: Motor and sensory function is normal.

As illustrated above, if the patient's sacral reflexes are intact on physical examination the SCI may be classified as incomplete, which is an early positive prognostic indication.

Whilst the ASIA physical examination is the most frequently used measure in the clinical setting, its limitations as a research tool include: 1. No tests of autonomic function;

2. Sensory classifications are subjective, and not testable in some patients, (ventilator-dependent, coma, cognitive/mood states, associated nerve injury);

3. Absence of key motor points between Cl-3 (inclusive) and T2-L1 (inclusive), also not testable (as above).

The ASIA and other, functional methods of injury classification (Ditunno et al 2000, Ditunno et al 2001, Stineman et al 1996, Marino et al 1999) can be prone to inter- observer error. In addition the confidence interval of the ASIA score has been demonstrated to be quite low, in part due to inter-rater reliability (Priebe et al 1992). Importantly the prognostic precision of physical examination can be extremely low, although early ASIA scores are used in prognostic assessment.

Motor recovery is categorised in two periods, "early" and "late". Early motor recovery might appear within 1 to 2 months of injury and is a positive prognostic sign (Ditunno et al 2000). A finite period for the appearance of spontaneous motor recovery is seen as ending at about 2 years from SCI diagnosis. Late recovery, after 2 years has been reported. Peripheral nerve regeneration in the "zone of injury", that is; one or two segments above and/or below the lesion, is the most frequently reported mechanism of late recovery. This late recovery mechanism is most commonly seen in patients with tetraplegia, where it is important functionally. Late recovery also can occur cases of paraplegia (in conus medullaris or cauda equina lesions).

The prognosis for motor recovery is very difficult to ascertain, particularly in the early SCI period. The techniques used are physical examination (for which objectivity is attempted using standardized scoring systems such as ASIA), neuro-imaging techniques (such as Magnetic Resonance Imaging [MRI]) and electrophysiological evaluation (Electromyography E.m.g.). For research purposes E.m.g. may be performed in conjunction with Transcranial Magnetic Stimulation (TMS). All these techniques are, however, primarily diagnostic and not prognostic and have received variable acceptance in the clinical setting. Particularly the physical examination regimes are relatively subjective and whilst they do have some prognostic capacity, in the early period this is not a highly sensitive test for prediction of motor recovery.

Early prognosis is important to the functional outcome of a SCI victim because certain treatment regimes may be administered to enhance an individuals' potential for motor recovery. Additionally from a psychological point of view the degree of loss of function is a paramount issue for socio-emotional adjustment.

SUMMARY OF THE INVENTION This invention arises from an investigation into variation of early perturbation of markers associated with bone remodelling following SCI which led to the discovery that levels of certain of these markers can be used to discriminate the potential outcomes for motor recovery in acute SCI patients.

In particular the data presented shows that SCI patients' prognosis for motor recovery can be discriminated by determining the degree of early bone resorption as expressed by associated markers. Hence markers associated with bone resorption in SCI can be described as surrogate indices of neurological prognosis.

In a broad form of a first aspect this invention might therefore be said to reside in a method of predicting chronic loss of function for a patient presenting with a central nervous system neuropathology said method including the steps of sampling a body fluid or tissue; assaying the body fluid or tissue quantitatively for one or more markers associated with a physiological function selected from the group consisting of bone resorption, remodelling or formation, a proinflammatory marker and an autoimmune antibody, and; comparing the result of the assay against a standard or predetermined value to make a prediction of the degree of chronic loss of function.

Loss of function might be defined by measurement as an ASIA score of AB as compared to CD, respectively to complete and incomplete or by other physical or neurological methods. From a histopathological perspective, spinal cord injury has been described as a "white matter injury". An ASIA AB (motor complete) lesion involves both the white (longitudinally oriented myelinated tracts) and grey matter neuronal bodies of the cord at the level of the lesion, (although a non-functional perimeter of white matter may be preserved, ie; classified as discomplete on Electromyography [E.m.g]). In contrast, in ASIA CD groups (motor incomplete) either some or all of the longitudinally oriented white matter (myelinated tracts) are preserved at and below the level of the lesion. Therefore, other (behavioural and clinical) variables being equal during the early phase of SCI, the bimodal trends in patients' bone markers and Bone Mineral Density (BMD) can be explained by the degree of secondary degeneration to the cord (temporally, downstream of loss of auto-regulation, auto-destruction (loss of neurons and oligodendrocytes in the penumbra and remote from the injury site). Clinically, there is considerable overlap between necrotic and apoptotic mechanisms of cell death and secondary demyelination. Experimental evidence suggests that tissue damage induces a central autoimmune response (secondary to cell inflammatory response to the Central Nervous System [CNS] tissue damage. As explained above, SCI disrupts with a very critical time course, the orchestrated expression of many gene families leading to peripheral immune cell invasion (neutrophils, monocytes, T-lymphocytes) and stimulation of microglia, reactive astrocytes. Nuclear transcription factor kappa b (NFk- b) is a key regulator of the CNS inflammatory response to injury. In SCI patients it is likely that levels of pro-inflammatory mediators in the Cerebro-Spinal Fluid (CSF) system are elevated for a finite period after injury, which could explain the magnitude of the elevated levels of bone resorption markers in early SCI patients and the characteristic time to peak. Experimental evidence also has shown that pro-inflammatory

(lipopolysaccharide [LPS] stimulated pro-apoptotic cytokines, leptin hormone) and excitotoxic mediators (glutamate), when elevated centrally, induce rapid bone loss. Therefore, differences in the degree of the early perturbation to bone resorption markers identified in the data supporting this invention in SCI patients, could be explained by differences in concentrations of central pro-inflammatory or other mediators linked to neuro-inflammatory responses in these patients. The present study might be considered to have identified statistically significant trends in SCI data that indicate that early SCI bone loss is caused by an auto-immune response to neurotrauma, and injury-dependent interactions between immune and neuroendocrine systems. Therefore in addition to the bone resorption markers already identified by experimentation one might expect that osteoprotegerin (OPG) and other factors such as receptor-activated nuclear transcription factor Kappa-5 fRANK), and its cognate ligand receptor activated nuclear transcription factor Kappa-2? ligand (RANKL)- also known as osteoclast differentiation factor (ODF, TRANCE , and OPGL (osteoprotegerin ligand) - with either affinity for or induced by proinflammatory mediators, may be elevated in the sera of SCI patients and show bimodal trends in relation to injury severity. In vitro investigations have shown that osteoprotegerin (OPG), a glycoprotein expressed in membrane bound and soluble forms, inhibits osteoclastic bone resorption via actions as a decoy receptor for RANKL. OPG/RANKL interactions have been implicated in the pathophysiology of numerous osteopenic bone diseases (age-related and postmenopausal osteoporosis [oestrogen deficiency], glucocorticoid exposure, T-cell activation (eg rheumatoid arthritis) and skeletal malignancies (myeloma metastases). It is also well known that the overproduction of pro-inflammatory cytokines (IL-6, IL-1 alpha, TNF- alpha and TNF-beta) is a potent stimulus for osteoclast activation. Thus one might postulate the those SCI sufferers that will go on to having complete loss of function will have higher initial levels of soluble RANKL (sRANKL), and/or ratio of sRANKL:OPG or lower sOPG than those that will go on to have an incomplete loss of function. Similarly, leptin hormone has been correlated with bone loss, via its central actions and, its serum levels with bone mineral density (BMD). Thus those SCI sufferers that go on to having a complete loss of function will have elevated levels of serum leptin relative to those that go on to having an incomplete loss of function, potentially after correction for body fat.

Additionally serum levels of proinflammatory mediators may also show bimodal trends, as may auto-antibodies.

An alternative hypothesis is that the micro-architecture of spinal cord tissue is altered after injury (eg, the dimensions of the extracellular space and microenvironment therein) with implications for synaptic transmission and extra-synaptic transmission. In pathological conditions, mechanisms may include neuron-glial communication, transmitter crosstalk or spill-over, long term depression (LTD) and, long term potentiation (LTD) etc. In this model, spinal neuron discharge (depression or potentiation) may modulate the release of amines and neuropeptides by perivascular sympathetic and sensory nerve terminals in the bone compartment(s). (Neuroskeletal mechanisms have been implicated in stimulation of bone marrow adipose cell proliferation and impaired dendritic cell function, as reported by recent bone biopsy studies of SCI patients). It is therefore postulated that qualitative and quantitative differences in the bone (marrow) microenvironment may explain the bimodal trends seen in SCI patients' early bone resorption. Molecules or factors associated with these responses may also exhibit a bimodality at early stage SCI.

The bone resorption markers might be factors that play a role in bone biochemistry in particular in bone resorption or secondary factors the level of which varies with the degree of bone resorption. On the other hand markers for bone remodelling functions other than remodelling may also provide a suitable bimodality.

Markers might thus be concentrations of systemic hormones (parathyroid hormone [PTH] or PTH fragments, parathyroid hormone receptor protein (PTHrP), 25-OHVit D, 1- 25,2(OH)Vit Dand other molecular factors (for example osteoprotegerin [OPG], receptor activated nuclear transcription factor kappa b [RANK], its ligand [RANKL], glutamate, leptin, prostaglandin E2 [PGE2]) that have been associated with bone metabolism. Other local factors associated with bone metabolism (noradrenalin [NA], neuropeptide Y-1,2,4 [NPY- 1,2,4], vasoactive intestinal peptide [VIP], substance P [SP] calcitonin gene-related peptide [CGRP], neurokinin A [NKA]), may also show bimodality. Factors of broader interest include follicle stimulating hormone (FSH), luteinising hormone (LH), 17B estradiol (E2), testosterone, sex hormone binding globulins (SHBG), or an index or ratio, for example, Free Androgen Index (FAI) etc).

Markers may be deoxypyridinoline (Dpyr), pyridinoline (Pyr), N-terminal cross linking telopeptide of Type 1 collagen (NTx) and C-terminal crosslinking telopeptide of Type 1 collagen (Ctx) and tartrate-resistant acid phosphatase isoform-5b (TRACP-5b). Other measures that might be used in the assessment on their own or in combination with other markers include, total body calcium content (TBCC), fat mass, or bone mineral density (BMD) in regions or sites of interest (measured by Dual Energy X-ray Absorptiometry [DEXA]) or intraosseous fat (measured by functional Magnetic resonance imaging [MRI]).

Clinical biochemistry (total calcium, ionised calcium, phosphate levels, haemoglobin, albumin, protein) and clinical haematology (immune cell counts) may also be used on their own or in combination with other markers. Bone- specific formation markers including Osteocalcin (OC), bone alkaline phosphatase (BAP),may also be used in a ratio to other markers.

Proinflammatory cytokines including but not limited to interleukin 6 (IL-6, IL-1, alpha, IL-11) and tumour necrosis factor alpha (TNF- ) may additionally be used as markers, as may auto antibodies in particular specific for central nervous system neurons and associated parts, and perhaps directed against gangliosides and may in particular be GM gangliosides and more particularly GM1- ganglioside.

Generally it is preferred that combinations of markers are used, for example Pyr:OC, DPyr:OC, HOP (hydroxyproline):creatine, Pyπcreatine, DPyrxreatine,

OPG:RANKL(OPGL), OPG:Pyr (and/or Ctx, Ntx), OPG:DPyr (and/or Ctx, NTx) and RANKL:Pyr.

In one form the sampling of the body fluid or tissue is on or before 52 weeks of onset of the condition, preferably before 25 weeks more preferably before 20 weeks. At least one marker shows bimodality at 3 weeks however the bimodality may be present at 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5 or 4 days following onset of the injury.

It is found that the most effective sampling time is about 12 to 16 weeks post injury, however there is clear pressure to have a prognosis for the patient as early as possible. Preferably the sample is a body fluid selected from the group comprising blood and urine, however other body fluids present perhaps in smaller quantities might also be taken such as perhaps tears, oral secretion.

It is proposed that the finding of the bimodality of level of the marker associated with early bone remodelling or resorption in SCI will have a similar predictive capacity to certain other neurological injury. Thus bone loss has similarly been described in other neuropathologies such as TBI (traumatic brain injury), cerebrovascular accident (CVA, brain ischaemia [infarction] or haemorrhagic stroke) and the neurodegenerative disorders ( (Multiple Sclerosis [MS], Amyotropic Lateral Sclerosis [ALS], Parkinson's Disease [PD], Alzheimer's Disease [AD], or other dementias. Thus TBI has similar sequences of progression, auto-destruction a period of disrupted permeability of the blood brain barrier, infiltration of non-resident immune cells and secondary degeneration. In particular it is proposed that the method may be applicable to Central Nervous System (CNS) associated neuropathologies in particular acute neuropathologies that may have resulted from injury, infection, toxins, or the like.

The assessment may in addition to predicting functional outcome also be useful for providing early monitoring of interventions and therapies, or if a neuro-degenerative condition, as a screening tool for pre-clinical diagnosis. The assessment may also be useful for monitoring disease progression in pathological states.

For a better understanding specific embodiments of the invention will now be described with reference to examples and drawings wherein.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 : is graph showing the change in level of bone mineral density (BMD) in patients that go on to exhibit complete loss of motor function as compared to patients that go on the exhibit incomplete loss of motor function. The change of bone mineral density is measured at intervals extending to 104 weeks following onset of SCI. Figure 2 is a graph showing the changing levels of White Cell Counts (WCC) and neutrophils in patients at intervals extending to 104 weeks following onset of SCI. Figure 3 is a graph showing the change in the ratio of Deoxypyridinoline to Creatinine at intervals extending to 24 weeks from onset of injury by SCI outcome as measured at 2 years from onset.

Figure 4 is a graph showing the change in the ratio of Pyridinoline to Creatinine at intervals extending to 24 weeks from onset of injury by SCI outcome as measured at 2 years from onset.

Figure 5 is a graph showing the change in the level of Osteocalcin at intervals extending to 24 weeks from onset of injury by SCI outcome as measured at 2 years from onset.

Figure 6 is a graph showing preliminary data for serum PTH levels at 10 days from admission in vertebral fracture controls (ASIA E) and patients with vertebral fracture and SCI (AB vs CD as determined at admission).

DETAILED DESCRIPTION OF THE ILLUSTRATED AND EXEMPLIED

EMBODIMENTS OF THE INVENTION

Some abbreviations and acronyms used throughout this specification are set out below.

ASIA - American Spinal Index

BBB - Blood Brain Barrier

BMD - bone mineral density

CNS - central nervous system

C.S.F. - cerebrospinal fluid

CVA - cerebrovascular accident

DEXA - Dual Energy X-ray Absorptiometry Dpyr: Cr -Deoxypyridinoline creatinine ratio

HDL - high density lipoprotein

HO - heterotopic ossification i.v.- intravenous (clinical usage); intracerebroventricular (experimental usage)

LDL - low density lipoprotein

LEMS - lower extremity motor score

LIF - leukemia inhibitory factor mAb - monocloncal antibody

NA - noradrenaline

NASCIS 1-111 - National Acute Spinal Cord Injury Study 1-111

NPY - neuroactive peptide Y

NSAID - Non Steroidal Anti-inflammatory Drug

OB-R — leptin receptor

OC - osteocalcin

PGSN - preganglionic sympathetic neuron

PTH/PTHrP-PTHRl - Parathyroid Hormone /Parathyroid Receptor Protein - Parathyroid Receptor 1

Pyr:Cr - pyridinoline creatinine ratio

QCT - quantitative computerised tomography

SCI - spinal cord injury

S-Cr -serum creatinine

SNS - sympathetic nervous system

S-P -serum phosphorus

TBI - traumatic brain injury

TNF-alpha - tumor necrosis factor - alpha

VEGF - vascular endothelial growth factor

VIP - vasoactive intestinal peptide

WBC - white blood cells

RANK - receptor activated nuclear transcription factor kappa- b

RANKL - receptor Activated Nuclear Transcription Factor Kappa- b Ligand

UEMS - upper extremity motor score EXAMPLE 1

Longitudinal measures of body composition and bone metabolic activity after a significant spinal cord injury (SCI): A comparison by injury severity Subjects and Methods Subjects

Thirty five (35) hospitalised patients, thirty two (32) men and three (3) women who were diagnosed with clinically significant SCI of less than 3 weeks duration participated in the study. Patients who met study age (18-55) and health criteria were selected from a cohort of 256 consecutive patients who presented to the Spinal Injuries Unit of the Royal Adelaide Hospital, over a five year period (1997-2001). (Patients with co-morbid bone and joint disease, lower extremity fracture, a history of mediations known to affect bone metabolism or chronic systemic disease were excluded). A SCI was defined as a traumatic injury of the spinal cord, but not the cauda equina or conus. Each patient's SCI was classified by severity (ASIA A-D), during a routine physical examination within 48 hours of hospital admission (Stover et al, 1996). Patients were aged from 18- 48 years (mean 29.9 ±9.1 years) and prior to their hospitalisation all patients resided in South Australia or its environs. All participants were skeletally mature, and no patients were in the osteoporotic BMD range at admission, as determined by DEXA. Of the group, no one had received a course of oral cortico-steroids (where dosage of cortico-steroid is defined as greater than 5 mg prednisolone, daily for 3 months). All patients received a bolus dose of methylprednisolone (MP) and iv. infusion within 48 hours of admission (according to NASCIS III protocol). One subject, (EH) a 48 year old woman commenced hormone replacement therapy (HRT) and another (AR), a 21 year old woman, was prescribed an oral contraceptive, (mean age 36 ± 13.9), Two (2) of the four women patients were multiparous, one (1) nulliparous, and two (2) were peri- menopausal.

Patients' health and compliance with medical and study protocols was monitored throughout. Most participants were prescribed dietary supplements (Vit C, High Protein Diet, Alimentary Zinc, but not 25-Vit D, Calcium or Vitamin K). Concomitant medications (Clexane, Baclofen, Gabapentin, Norfloxican, Oxybutinin, Coloxyl, Senna etc) that frequently are used in SCI management also were monitored throughout (all patients were anti-coagulated in the initial weeks of admission). During rehabilitation all subjects participated in physical, occupational therapy and gym programs at levels commensurate with function and health. Some continued their physical therapy or training programmes after discharge. Lower extremity functional electrical stimulation (FES) programs were capped at a maximum exposure of 1.5 hours stimulation per limb per week.

At the conclusion of the study two (2) years from SCI diagnosis, patients completed physical (ASIA) and psychological outcome evaluations. Psychological tests were composed of the Quality of Life and Locus of Control Instrument (QUALY), Hospital and Anxiety and Depression Scale (HADS). Physical evaluation was comprised of: medical and physical examination, ASIA score, upper and lower extremity motor scores (UEMS, LEMS respectively) (Waters et al, 1991 'a, Water et al, 1997b) as well as the Functional Independence measure (FIM) (Uniform Data Systems Inc). Clinical biochemistry and haematology also were performed. Other, less tangible aspects of patient outcome, such as spasticity and chronic pain (Siddall et al, 1999) were determined by subjective means (patients self-rated perception of chronic pain using a visual analogue scale [VAS]).

During the course of the study, four (4) patients were withdrawn, two (2) voluntarily and two (2) for adherence to protocol. Six (6) patients developed secondary complications of bone and/or joint (all heterotopic ossification) and one (1) clinical but not endocrinological signs of hypercalcaemic crisis.

The study was approved by the Combined Research Ethics Committee of the Royal Adelaide Hospital and the University of Adelaide. All patients provided informed consent.

Methods Bone biochemistry was performed at 3 weeks and then repeated at 6, 12, 24 and 52 weeks from SCI diagnosis. All urine and blood specimens were obtained early morning (between 08.00 and 10.00am), after an overnight fast. Spot urine samples were collected at the second void. Venipuncture was performed in the upper limb. All specimens were handled and stored as recommended by Withold (1996). Analysis was performed at the Institute of Medical and Veterinary Science, (IMVS), Adelaide, South Australia. Levels of Pyr:Cr, Dpyr:Cr, and HOP:Cr were determined by the HPLC method and OC, using commercial kits. Clinical biochemistry and haematology tests also were performed at these intervals. All laboratory tests were repeated 2 years from SCI diagnosis, at the final clinical evaluation.

Densitometry (DEXA) was performed at 3 weeks from SCI diagnosis using a fan beam densitometer (Lunar Expert, Lunar Corp, Madison, Wisconsin, USA). The first scan was acquired by one of two operators (CS, PW). Subsequent scans were repeated at intervals of 6, 12, 24 and 52 weeks from SCI diagnosis. A final scan was performed at two (2) years from SCI diagnosis to correspond with clinical outcome evaluations. Software allowed for the derivation of body mass into three (3) tissue compartments (Fat, Lean and Bone, or BMD), and BMD in regions (arms, legs). Total Body Calcium or Bone Mineral Content (BMC) and Bone Mineral Density (BMD), are described as relative values (% change from initial baseline measure). Measurements also were taken of spine (L2-4) and hip sites: specifically the proximal femur, total femur, neck of femur (NOF), Ward's Triangle, greater trochanter and the inter-trochanteric regions.

Expert 1.92 software was used at all times. System precision was monitored on a weekly basis, using a Lunar densitometry spine phantom. Precision error was calculated as <1% coefficient of variation.

Statistical Methods

The "intention to treat principle" was applied to the statistical methods. Values are expressed as percentage (%) change, calculated thus:

BMD = BMD Tx- BMD TO/ BMD To.100, where To is the value for the baseline three week scan. For FM, % change was calculated as a percent of total body mass at baseline, using the formula:

FM = FM Tx - Mb To/ Mb To.100 where Mb, is body weight (not fat mass)

Biochemical data are presented in absolute values or, as a ratio to creatinine (means, SD). A sliding dichotomy was introduced, where the point of dichotomisation was tailored to the baseline prognosis. Data were separated into bands, of severe (ASIA A-B) moderate (ASIA C-D) and mild injury (ASIA E). The Yes/No motor recovery, high/low dichotomy, was explored using patient's final outcome data (ASIA scores at two (2) years from SCI diagnosis). All ASIA scores were assessed by the same medical officer (MJ). Group membership, was applied as (a) motor complete (ASIA A and B) or (b) motor incomplete (ASIA C or D) or normal E). Student T tests then were performed (two tailed, unequal variance), to compare trends by diagnosis, as well as group trends by injury severity across time and at individual time points. A probability value p< 0.05 was considered statistically significant.

Densitometry data also were segregated according to (1) impairment group, [tetraplegia (C2-T1) paraplegia (T2- T12)], (2) ASIA score [A-B, C, D-E] (3) Level [above T6 and T7 -T12]. Curves were generated across time for each parameter and regression equations fitted. Pearson's correlations also were applied to BMD and FM at individual time points.

Results

A summary of the results are shown in table 1. Table 1 : Serum and urine bone biochemistry and clinical haematology shown by injury severity.

SCI Duration 3 (wks) 6 (wks) 12 (wks) 24 (wks)

Variable mean,(SD) p value mean,(SD) p value mean.(SD) p value mean,(SD) p value (by ASIA outcome) A-B C-D A-B C-D A-B C-D A-B C-D

PyπCr 299 (111) 201(99) .02 ^: 514 (291) 262(101) .01 ** 618(339) 277(101) .0001*** 237(76) 181(86) .002** (nmol.mmol'^l) 74(26) 55.9 (24) >.05 114(50) 74(24) .01* 132(58) 65(15) .001*** 86(33) 46(23) .01**

Dpyr : Cr (nmol.mmol^'1* 396 (157) 389 (181) >.05. 648(356) 444(399) .02* 544 (209) 339 (149) >0.05 337(181) 305(185) .01**

HOP:Cr

(nmol.mmol ^~1} > 1.5(0.2) 1.4(0.2) >.05 1.5 (0.2) 1.7 (0.9) >.05 1.5 (0.01) 1.3 (0.1) .01* 1.5 (0.01) 1.2 (0.2) .OP

Phospate (mmolL^'V 2.3(0.1) 2.3(0.11) >.05 2.4 (0.1) 2.3 (0.2) >.05 2.4(0.1) 2.4(0.1) >0.05 2.4(0.1) 2.3(0.1) .01**

Total Calcium (mmol :L ^~l> 63 (10) 64(4.0) >.05 66 (0.1) 63 (2.4) .07 68 (7) 69(1.4) >0.05 69(5.5) 69(4) >0.05

Protein (g.L-» 0.6 (0.2) 0.5 (0.2) >.05 1.4(2) 0.5 (0.3) .06* 0.5 (0.2) 0.7(0.2) <0.05* 0.5(0.2) 0.2(0.2) >0.05

Monocytes (xlC.Lr" 121(17.5) 132(8.5) .06 119(42) 119(48) >.05 114(51) 143(9) >0.05 124(35) 119(47) >0.05

Haemoglobin (g.L-'> >.05 >.05 >0.05 >0.05

Osteocalcin

The above experiments have shown that Pyr:Cr, DpyπCr, HOP, se-Ca, and se-Phos) are explanatory variables for SCI patients' motor outcome. As demonstrated by levels of markers of bone resorption (in serum and urine) grouped by patient outcome (dichotomised into Motor Recovery (CD), No Motor Recovery groups(AB)), levels of bone-specific markers can discriminate patients with potential for motor recovery. The statistical trends were seen consistently across time, (Pyr, Dpyr, HOP) and across all markers of bone resorption, as observed on at least one and usually more individual time points (Pyr, Dpyr, HOP, se-Ca, se-Ca, se-Phos). Markers of bone formation (OC) did not show early trends, unless patients developed peri-articular bone ossifications. Thus bone-specific resorption markers showed sensitivity to differentiate an individual patient's prognosis after SCI. In contrast bone formation markers showed a correlation to risk for bone complication, but not to SCI recovery trends.

Other early trends included elevated immune cell counts in the systemic circulation (WCC), which points to pro inflammatory cytokine activation. The trends in patients' early bone biochemistry were subsequently confirmed by a decline in BMD, as shown by DEXA. DEXA, as used in the present study, has proven reliability for BMD measurement in the ambulant reference population with a confidence interval of 1 %. Interpretation of densitometry data from medically fragile patients diagnosed with SCI is challenging. The % change to SCI patients' bone mass (up to 37% decline in hip BMD in the first 2 years) is strongly correlated with the magnitude of the peak amplitude of excretion of bone resorption markers. Additionally, the BMD slope is correlated with the rise to peak amplitude (slope) in bone resorption markers. Unfortunately, due to well known delay between the appearance of heightened bone metabolic activity and osteoid formation, DEXA scans have diagnostic value, but no real value from a prognostic perspective. However, the rather unusual relationship between fat mass, BMD and injury severity in these patients may indicate that Functional Magnetic Resonance Imaging in bone marrow, (f-MRI) or other measures of bone marrow cellularity, have potential to differentiate early differentiation plasticity in resident bone marrow cells, also correlated to injury severity. In summary, the study, has demonstrated that bone-specific biochemistry is an explanatory variable for SCI patients' neurological outcome. The simple bone biochemistry assays performed during the study, showed predictive precision within 3- 12 weeks of patients' diagnosis. A power analysis revealed a 100 % confidence interval at the 12 week point to predict membership of the "No Motor Recovery" ASIA AB group. Statistically, the early trends in SCI bone biochemistry indicate potential to discriminate potential for recovery.

Table 2: *Summary of Pilot Data at Visit 1 (10 days from hospital admission). Control SCI SCI Comments (ASIA E) (ASIA C- (ASIA A- D) B)

Variable Fracture Fracture Fracture vertebral vertebral vertebral

Dpyr Cr 29 57 70 *Suppressed in SCI patients by injury

(nmol.mmol^'1) severity see Table 1

Pyr :Cr 107 238 291 *Suppressed in SCI patients by injury

(nmol.mmol^'1) severity see Table 1

Osteocalcin 4.9 2.7 19.2 -

PTH 2.0 1.2 0.8 *Trend- suppressed in SCI patients by

(pmol.L ^~}) injury severity

25 OH-Vit D 86 68 25 *Trend- suppressed in SCI patients by

(nmol.L ^') injury severity l-25,2OH-Vit 56 148 26 No trend

D (pmolL -¹) hGH 0.6 8.0 0.9 No trend

(mlU.L¹)

IGF-1 58 45 44 Trends between controls and SCI

(nmol.L ) patients (suppressed in SCI) but not by injury severity

Estradiol 73 105 73 No trend

(pmoLL'¹)

Testosterone 18.9 2.4 6.4 Trends between controls and SCI

(nmol.L ^~J) patients (suppressed in SCI) but not by injury severity

Free 0.38 0.07 0.2- 0.6 Suppressed in both SCI diagnostic

Testosterone groups

(nmol.L )

FAI 52.3 20.0 58.2 No trend

(%)

SHBG 36 12 11 Trend between Control and SCI, not

(nmol.L ) by SCI severity (suppressed in SCI diagnostic group)

TSH 1.2 0.8 1.4 No trend

(mlU.L¹)

Free T4 15 14 15 No trend

(pmol.L ^'')

Total - 0.9 1.3 ?

Triglycerides

(nmolL ^'')

Total 4.3 4.0 3.4 ?

Cholesterol (nmol.L )

HDL 1.4 0.8 0.7 Trend between Control and SCI, not

Cholesterol by SCI severity

(nmol.L

LDL 2.3 2.8 2.1 No trend

Cholesterol

(nmol.L ^'')

Glucose - - 4.5 -

(nmol.L ^~])

Albumin 52 33 32 Trend between Control and SCI, not

(g-L-¹ ) by SCI severity

Haemoglobin 159 118 114 Trend between Control and SCI, not

(g- ^'1) by SCI severity

RBC 5.28 3.88 3.94 No trend

(x 10 ⁿ. ¹)

PCV 0.45 0.36 0.36 No trend

(L.L-¹)

WCC 6.42 8.75 11.3 Trend -elevated by SCI severity

(x 10⁹.L-^])

Neutrophils 3.88 5.93 8.25 *Trend -elevated by SCI severity

(x 10 L^'1)

Ionized Ca 2.55 1.17 1.34 No trend

(mmol.L )

Total Ca 1.22 2.25 2.47 Differences between Control and SCI

(mmolL ^~]) patients , but not differences by injury severity

Phosphate 1.51 1.23 1.62 No trend

(mmoLL ^~])

* Data for previously healthy men (age range 20-39 yrs) admitted to the RAH between June and August 2004

It is now postulated that other factors associated with bone metabolism may show additional sensitivity to predict outcome. However, a simple 2x2 cross-tab analysis can reduce sensitivity to other variables. Table 2 shows preliminary data for calciotropic hormones and sex steroids at 10 days from admission in vertebral fracture controls (ASIA E) and patients with vertebral fracture and SCI (AB vs CD) as determined at admission. Further analysis of trends in fracture controls vs patients with SCI was inappropriate.

EXAMPLE 2 Further factors that can be taken as surrogate indicator for prognosis of motor recovery in

SCI patients. In view of trends shown in the data presented in example 1, it is proposed that other markers that have been demonstrated either in vitro or in vivo, to participate in bone remodelling can also be used. Specifically, the next phase of study will be geared to determine whether such factors, can discriminate motor complete from motor incomplete.

Factors of specific interest include:

Pyridinoline (Pyr), Deoxy Pyridinoline (D-Pyr) and C-terminus cross-linked telopeptide of Type 1 collagen (Ctx) and N-terminus cross-linked telopeptide of Type 1 collagen

(NTx) . Osteoprotegerin (OPG),

Receptor activated nuclear transcription factor -kB (RANK),

RANKL (L=ligand),

Leptin hormone ,

Calcitonin (CT)/Calcitonin Gene related peptide (CGRP) as well as Parathyroid hormone (PTH), its fragments, parathyroid hormone receptor proteins

(PTHrP), Growth Hormone (GH), insulin-like growth factor binding proteins 1 - 3 (IGF-

BP1- 3) and sex steroids (testosterone (Free Androgen Index -[FAI]), sex hormone binding globulin [SHBG}, 17b estradiol (E2), progesterone etc as described above). Furthermore, analysis of outcome measures will be composed of single factors, and ratios, eg. Pyr: OPG Dpyr: OPG OPG: RANL OPG: RANKL OPG: LEPTIN

Subjects - Selection and exclusion criteria Inclusion

ASCI Subjects

Inpatients of the Acute Spinal Injuries Unit, Royal Adelaide Hospital.

Men and women, aged 18 years and over.

SCI (ASIA A-D), with or without vertebral fracture*, either conservative or surgical stabilisation.

C2 segment to cauda equina (provided they are not ventilator dependant at the time of study commencement).

Within 10 days of acute hospital admission.

Clinical Controls

Inpatients of the Orthopaedic Wards, Royal Adelaide Hospital.

Men and women aged 18 years and over.

Acute vertebral fracture, no neurological loss (ASIA E).

Within 10 days of acute hospital admission.

Exclusion -

Ventilator-dependent patients (precludes DEXA measures).

Active diseases/disorders of bone metabolism.

History of/or recent exposure to agents or hormonal therapies known to influence bone metabolic activity (cortico-steroids, cyclosporins, Hormone Replacement Therapy

(HRT), oral contraceptives, etc).

Active systemic infection, eg, transmissible viral disease(s). Neoplasm, active primary or secondary metastases or, Hx of cancer or, treatments for cancer (radiotherapy, chemotherapy, SERMS (specific estrogen receptor modulators).

Precautions - Medical fragility related to ASCI, acute complications of ASCI.

Study Plan and Design Serological indices of bone formation (Osteocalcin, Bone Alkaline Phosphatase [BAP]) and resorption (C-telopeptide of Type 1 collagen [Ctx]), will be determined at less than 3 weeks, 3, 6, 12, 24, and 52 weeks from hospital admission. Specimens will be analysed at the IMVS laboratories, Royal Adelaide Hospital, or other participating centres, using standardised laboratory protocols. Osteoprotegerin (OPG), RANK, RANKL, leptin and LEPHR levels will be analysed by DOTS laboratories, using ELISA and other technologies. Serum will be stored in liquid nitrogen and ELISA and other assays performed periodically.

Renal excretion of markers of bone resorption (Deoxypyridinoline, Pyridinoline) will be determined as a ratio to creatinine. Urine specimens will be obtained at less than 3 weeks, 3, 6, 12, 24 and 52 weeks from hospital admission. IMVS or other laboratories will perform routine assays. Enzyme linked immunosorbent assays (ELISA's) or other assays may be performed.

Serum concentrations of growth hormone (GH), insulin-like growth factor-1 (IGF-1, IGFBP1-3), thyroid stimulating hormone (TSH), parathyroid hormone (PTH), 25-OHVit D, lalpha,25(OH)₂D₃, leptin hormone (LH, LEPHR) and sex steroids (Free Androgen Index, estradiol- E2 etc) will be assayed by the IMVS. (Prostaglandin E2 (PGE2) concentrations will be determined in urine).

Protocols require collection of specimens after an overnight fast (from midnight), no other exclusions will apply.

Compete blood evaluation (CBE) will be performed as indicated clinically. Haematology and biochemistry will be evaluated at identified sample points. As carbohydrate intolerance is prevalent in chronic SCI patients (Baumann et al 1996), a standard 75g Oral Glucose Tolerance Test (OGTT) will be performed at 12 months, or before this time, if indicated clinically.

Densitometric evaluation (Dual Energy X-ray Absorptiometry [DEXA], Lunar Prodigy, Lunar Corp, Madison, Wisconsin), will be performed either less than/or at 3 weeks and repeated at 6, 12, 24 and 52 weeks from hospital admission. Scans will be acquired by the Dept Nuclear Medicine, Royal Adelaide Hospital or, in consultation with the RAH, by other participating centre(s). System precision will be ascertained on a weekly basis, and confirmed by each participating centre. The Dept Nuclear Medicine will collate, and analyse all radiological data.

Clinical and functional outcome measures will be composed of: American Spinal Injuries Association Score (ASIA). [Consortium for Spinal Cord

Medicine, Clinical Practice Guidelines (1999)].

Functional Independence Measure (FIM, Uniform Data Set etc).

Walking Index of SCI- WISCI [Ditunno et al (2001)].

Upper and Lower Extremity Motor Scores -UEMS, LEMS [Waters et al 1997a, 1997b]). Independent medical examiners who are blinded to the study protocol will administer

ASIA physical examinations. A neuro-physiotherapist will perform other physical tests, as defined in the protocol.

Outcomes Statistical analysis of study outcome measures (endocrinological, densitometric, bone biochemical, clinical/physical) will comprise: parametric (descriptive statistics, means, SD, students' t-test (alpha level 5%), Pearson's correlation coefficients). Non-parametric tests will be applied when appropriate (Spearman's ranked correlation coefficients to ranked ASIA data). The Mann- Whitney Test will be applied during subgroup analyses. In addition to standard statistical methods, ie ratios, thresholds appropriate to data treatment etc, data may be modelled to Bayesian or other algorithmic formulae, as determined by a biostatistician. Various features of the invention have been particularly shown and described in connection with the exemplified embodiments of the invention, however, it must be understood that these particular arrangements merely illustrate and that the invention is not limited thereto and can include various modifications falling within the spirit and scope of the invention.

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Claims

1. A method of predicting chronic loss of function for a patient presenting with a central nervous system neuropathology said method including the steps of sampling a body fluid or tissue, assaying the body fluid or tissue quantitatively for one or more markers associated with a physiological function selected from the group consisting of bone biochemistry including bone resorption and remodelling, pro and anti inflammatory cytokines, densitormetry , haemotology, clinical biochemistry, calcium regulatory hormones, endocrinology, gonadal steroids, a proinflammatory marker and an autoimmune antibody, and comparing the result of the assay against a standard or predetermined value to make a prediction of the degree of chronic loss of function.

2. A method of predicting chronic loss of function as in claim 1 wherein the marker is selected from group consisting of Dpyr (deoxypyridinoline), pyr (pyridinoline), Cr (creatine), HOP (hydroxyproline), osteocalcin (OC), bone alkaline phosphatase (BAP)osteoprotegerin (OPG), RANK, RANKL, Leptin, C- terminal cross- linking telopeptide of Type 1 collagen (Ctx), N- terminal cross- linking telopeptide of Type 1 collagen NTx, interleukin-1 alpha (IL-1 alpha), interleukin-6 (IL-6) interleukin-11 (IX- 11), tumour necrosis factor-alpha (TNF-alpha), tumour necrosis factor-b (TNF- b), bone density, total body calcium content (TBCC), fat mass, regional or site-specific BMD, inflammatory and red cell counts, total calcium, ionised calcium, phosphate levels, haemoglobin, albumin, parathyroid hormone (PTH), 25-Vit D, 1-25 (OH)2D3, prostaglandin E2 (PGE2); growth hormone (hGH), or GH releasing hormone (GHRH), insulin-like growth hormone- 1 (IGF-1), insulin-like growth factor binding protein 1 (IGF-BP1, insulin-like growth factor binding protein 2 (IGF-BP2), insulin-like growth factor binding protein 1 (IGF-BP 3), leptin hormone, calcitonin (CT), calcitonin gene- related peptide (CGRP), parathyroid hormone (PTH), testosterone, free testosterone, Free Androgen Index (FAI), 17-b- oestradial (E2) , prolactin, progesterone, follicle- stimulating hormone (FSH), luteinising hormone (LH), autoantibodies directed to central nervous system neurons, autoantibodies directed against gangliosides, or immunoglobulin serum auto antibodies (AAb)-IgG and IgM AAbs to neurofϊlament proteins, alpha internexin (alpha-INX).

3. A method of predicting chronic loss of function as in either claim 1 or 2 wherein two or more makers are assayed and the relative proportions of the markers are compared to the standard or predetermined value.

4. A method of predicting chronic loss of function as in claim 1 wherein the marker is a marker for bone resoφtion.

5. A method of predicting chronic loss of function as in claim 4 wherein the bone resorption marker is selected from the group consisting of Dpyr, pyr, Cr, HOP, OC, BAP, OPG, RANK, RANKL, Leptin. Ctx and Ntx.

6. A method of predicting chronic loss of function as in claim 4 wherein the bone resorption marker is selected from the group consisting of Dpyr, Pyr, Cr and HOP.

7. A method of predicting chronic loss of function as in claim 4 wherein levels of two or more markers is assayed and the relative proportions of the markers are compared to the standard or predetermined value.

8. A method of predicting chronic loss of function as in claim 8 wherein the bone resorption marker is selected from the group consisting of Dpyr, Pyr, Cr and HOP.

9. A method of predicting chronic loss of function as in claim 8 wherein level of two or more markers are measured and a value is calculated from the relative proportion of two markers selected from the group consisting of PyπOC, DPyr:OC, HOP:Cr, Pyr:Cr, DPyr:Cr, OPG:RANKL, OPG:Pyr, OPG:Ctx, OPG:Pyr:Ctx, OPG:DPyr, OPG:DPyr:Ctx. PyπCtx and RANKL:Pyr, and said relative proportion is compared to the standard or predetermined value.

10. A method of predicting chronic loss of function as in claim 8 wherein level of two or more markers are measured and a value is calculated from the relative proportion of two markers selected from the group consisting of Pyr:Cr, DPyπCr and HOP:Cr and said relative proportion is compared to the standard or predetermined value.

11. A method of predicting chronic loss of function as in claim 1 wherein the marker is a proinflammatory marker.

12. A method of predicting chronic loss of function as in claim 11 wherein the marker is selected from the group consisting of leptin hormone, gonadal steroids (17-b estradiol

[E2], progesterone, testosterone, prolactin, follicle stimulating hormone [FSH], luteinising hormone [LH]), proinflammatory cytokines, (interleukin-6 [11-6], interleukin- 11 [11-11], interleukin-1 alpha (11-1), tumour necrosis factor alpha, beta [TNF-alpha, beta].

13. A method of predicting chronic loss of function as in claim 1 wherein the marker is associated with bone formation or remodelling or bone micro architecture.

14. A method of predicting chronic loss of function as in claim 13 wherein the marker is selected from the group comprising densitometric evaluation of total body calcium content (TBCC), regional or site-specific BMD; serum or urine biochemistry and bone- specific biochemistry serum total calcium, serum ionised calcium, serum phosphate levels, serum osteocalcin (OC), bone alkaline phosphatase (BAP), the level of calcium regulatory hormones GH/IGF-1, PTH, VitD3, PGE2, calcitonin, calcitonin gene-related peptide (CGRP), insulin-like growth factor and its binding proteins 1-3., or bone micro- architecture.

15. A method of predicting chronic loss of function as in claim 1 wherein the marker is associated with bone micro architecture.

16. A method of predicting chronic loss of function as in claim 15 wherein the bone micro-architecture is measured either by micro quantitative computerised tomography [QCT] orhistomorphometrically.

17. A method of predicting chronic loss of function as in claim 1 wherein the marker is a pro or anti inflarnmatory cytokine.

18. A method of predicting chronic loss of function as in claim 17 wherein the marker is selected from the group consisting of IL-lalpha, IL-6, IL-11, TNF-alpha and TNF- b.

19. A method of predicting chronic loss of function as in claim 1 wherein the marker is a densitometry measure.

20. A method of predicting chronic loss of function as in claim 19 where the densitometry measure is selected from the group consisting of total body calcium content (TBCC), fat mass, regional or site-specific BMD.

21. A method of predicting chronic loss of function as in claim 1 wherein the marker is selected from the group consisting of total calcium, ionised calcium, phosphate levels, haemoglobin and albumin.

22. A method of predicting chronic loss of function as in claim 1 wherein the marker is an endocrinological marker.

23. A method of predicting chronic loss of function as in claim 22 wherein the endocrinological marker is selected from the group consisting of PTH, testosterone, free testosterone, FAI, E2 , prolactin, progesterone, FSH and LH.

24. A method of predicting chronic loss of function as in claim 1 wherein the marker is the presence of an autoantibody.

25. A method of predicting chronic loss of function as in claim 25 wherein the autoantibody is selected from the group consisting of autoantibodies directed to central nervous system neurons, autoantibodies directed against gangliosides, or immunoglobulin serum auto antibodies including to IgG and IgM, autoantibodies to neurofilament proteins and autoantibodies to alpha internexin (alpha-INX).

26. A method of predicting chronic loss of function as in claim 1 wherein the condition is selected from the group comprising spinal cord injury (SCI), traumatic brain injury (TBI), cerebrovascular accident (CVA), either ischemic and haemorrhagic stroke), multiple sclerosis (MS), Alzheimer's Disease (AD), Parkinson's Disease (PD), amyotrophic lateral sclerosis (ALS-also known as "Lou Gehrig's" Disease), and emergent diseases (including human immunodeficiency virus (HIV)-AIDS, bovine spongiform encephalitis (BSE), Creuztfeld Jacob Disease (CJD).

27. A method of predicting chronic loss of function as in any one of claims 1, 4 , 9 or 10 wherein the condition is SCI.

28. A method of predicting chronic loss of function as in either claim 1 or 27 wherein the sampling is taken between 3 and 25 weeks post injury.

29. A method of predicting chronic loss of function as in either claim 1 or 27 wherein the sample is taken 3 weeks post injury or later.

30. A method of predicting chronic loss of function as in claim 29 wherein the sample is taken about 12 to 16 weeks post injury.

31. A method of predicting chronic loss of function as in claim 1 or 27 wherein the body fluid or tissue is a non cerebrospinal fluid (CSF) body fluid.

32. A method of predicting chronic loss of function as in claim 31 wherein the body fluid is blood or urine.

33. A method of predicting chronic loss of function as in claim 27 wherein the loss of function is at 1 year post injury.

34. A method of predicting chronic loss of function as in claim 27 wherein the loss of function predicted is at 2 years post injury.

35. A method of predicting chronic loss of function as in claim 27 wherein the distinction of loss of function is complete vs incomplete.

36. A method of predicting chronic loss of function as in claim 35 wherein loss of function is as determined by the ASIA score CD as compared to a score of AB.

37. A method of predicting chronic loss of function for a patient presenting with a central nervous system neuropathology said method including the steps of sampling a body fluid or tissue, assaying the body fluid or tissue quantitatively for one or more markers from the group consisting of pyr, Dpyr, Ctx, Ntx, PTH 25-Vit D, l-25(OH)2D3, and comparing the result of the assay against a standard or predetermined value to make a prediction of the degree of chronic loss of function.

38. A method of predicting chronic loss of function as in claim 37 wherein two or more markers are assayed and the relative proportions of the two or more markers are compared to the standard or predetermined value.