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Multiple sclerosis (OMIM 126200) is a common disease of the central nervous system in which the interplay between inflammatory and neurodegenerative processes typically results in intermittent neurological disturbance followed by progressive accumulation of disability.1 Epidemiological studies have shown that genetic factors are primarily responsible for the substantially increased frequency of the disease seen in the relatives of affected individuals;2,3 and systematic attempts to identify linkage in multiplex families have confirmed that variation within the Major Histocompatibility Complex (MHC) exerts the greatest individual effect on risk.4 Modestly powered Genome-Wide Association Studies (GWAS)5-10 have enabled more than 20 additional risk loci to be identified and have shown that multiple variants exerting modest individual effects play a key role in disease susceptibility.11 Most of the genetic architecture underlying susceptibility to the disease remains to be defined and is anticipated to require the analysis of sample sizes that are beyond the numbers currently available to individual research groups. In a collaborative GWAS involving 9772 cases of European descent collected by 23 research groups working in 15 different countries, we have replicated almost all of the previously suggested associations and identified at least a further 29 novel susceptibility loci. Within the MHC we have refined the identity of the DRB1 risk alleles and confirmed that variation in the HLA-A gene underlies the independent protective effect attributable to the Class I region. Immunologically relevant genes are significantly over-represented amongst those mapping close to the identified loci and particularly implicate T helper cell differentiation in the pathogenesis of multiple sclerosis.

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Multiple sclerosis is characterized by inflammatory demyelination and irreversible axonal injury leading to permanent neurological disabilities. Diffusion tensor imaging demonstrates an improved capability over standard magnetic resonance imaging to differentiate axon from myelin pathologies. However, the increased cellularity and vasogenic oedema associated with inflammation cannot be detected or separated from axon/myelin injury by diffusion tensor imaging, limiting its clinical applications. A novel diffusion basis spectrum imaging, capable of characterizing water diffusion properties associated with axon/myelin injury and inflammation, was developed to quantitatively reveal white matter pathologies in central nervous system disorders. Tissue phantoms made of normal fixed mouse trigeminal nerves juxtaposed with and without gel were employed to demonstrate the feasibility of diffusion basis spectrum imaging to quantify baseline cellularity in the absence and presence of vasogenic oedema. Following the phantom studies, in vivo diffusion basis spectrum imaging and diffusion tensor imaging with immunohistochemistry validation were performed on the corpus callosum of cuprizone treated mice. Results demonstrate that in vivo diffusion basis spectrum imaging can effectively separate the confounding effects of increased cellularity and/or grey matter contamination, allowing successful detection of immunohistochemistry confirmed axonal injury and/or demyelination in middle and rostral corpus callosum that were missed by diffusion tensor imaging. In addition, diffusion basis spectrum imaging-derived cellularity strongly correlated with numbers of cell nuclei determined using immunohistochemistry. Our findings suggest that diffusion basis spectrum imaging has great potential to provide non-invasive biomarkers for neuroinflammation, axonal injury and demyelination coexisting in multiple sclerosis.

The dissociation between magnetic resonance imaging (MRI) and permanent disability in multiple sclerosis (MS), termed the clinicoradiological paradox, can primarily be attributed to the lack of specificity of conventional, relaxivity-based MRI measurements in detecting axonal damage, the primary pathological correlate of long-term impairment in MS. Diffusion tensor imaging (DTI) has shown promise in specifically detecting axonal damage and demyelination in MS and its animal model, experimental autoimmune encephalomyelitis (EAE). To quantify the specificity of DTI in detecting axonal injury, in vivo DTI maps from the spinal cords of mice with EAE and quantitative histological maps were both registered to a common space. A pixelwise correlation analysis between DTI parameters, histological metrics, and EAE scores revealed a significant correlation between the water diffusion parallel to the white matter fibers, or axial diffusivity, and EAE score. Furthermore, axial diffusivity was the primary correlate of quantitative staining for neurofilaments (SMI31), markers of axonal integrity. Both axial diffusivity and neurofilament staining were decreased throughout the entire white matter, not solely within the demyelinated lesions seen in EAE. In contrast, although anisotropy was significantly correlated with EAE score, it was not correlated with axonal damage. The results demonstrate a strong, quantitative relationship between axial diffusivity and axonal damage and show that anisotropy is not specific for axonal damage after inflammatory demyelination.

Previously, we tested the prediction that axonal damage results in decreased axial diffusivity ( ʈ ) while demyelination leads to increased radial diffusivity ( Ќ ). Cuprizone treatment of C57BL/6 mice was a highly reproducible model of CNS white matter demyelination and remyelination affecting the corpus callosum (CC). In the present study, six C57BL/6 male mice were fed 0.2% cuprizone for 12 weeks followed by 12 weeks of recovery on normal chow. The control mice were fed normal chow and imaged in parallel. Biweekly in vivo DTI examinations showed transient decrease of ʈ in CC at 2-6 weeks of cuprizone treatment. Immunostaining for nonphosphorylated neurofilaments demonstrated corresponding axonal damage at 4 weeks of treatment. Significant demyelination was evident from loss of Luxol fast blue staining at 6 -12 weeks of cuprizone ingestion and was paralleled by increased Ќ values, followed by partial normalization during the remyelination phase Diverse central nervous system (CNS) disorders involve white matter pathology leading to myelin and axon dysfunction (1-6). However, current neurologic examinations are not capable of differentiating the underlying axon and myelin pathologies causing the deficits (7,8). Recently, an analytical approach interpreting magnetic resonance diffusion tensor imaging (DTI) data in light of white matter pathology has been proposed (9 -11). Briefly, directional diffusivities of water molecules in white matter derived from DTI are separated into two components, i.e., axial ( ʈ ) and radial ( Ќ ) diffusivities describing water diffusion along and across axonal tracts, respectively. It has been demonstrated that axonal injury, such as axonal swelling and Wallerian degeneration (10), results in reduced ʈ , while dysmyelination increases Ќ without changing ʈ (11).The development of an effective therapy targeted at repairing axons and the protective myelin sheath may benefit from improved diagnostic tools that are capable of detecting and differentiating axonal damage and demyelination in various CNS disorders. To validate the aforementioned DTI parameters as surrogate markers of axon and myelin injury, the well-characterized CNS demyelination and remyelination mouse model of cuprizone (bis-cyclohexanone oxaldihydrazone) ingestion has been examined serially in living mice in the present study. Cuprizone, 0.2% by weight, was mixed in ground rodent chow and fed to a group of male C57BL/6 mice. The corpus callosum (CC) is the only tract in this mouse model that has been consistently documented to undergo massive and unequivocal demyelination as a result of cuprizone toxicity and remyelination after removal of cuprizone from the feed (12-16). The myelin and axonal pathology of the CC in the cuprizone-treated mice was demonstrated by histopathology and correlated with ex vivo DTI (9). Thus, the CC is the target white matter tract of the current study to test the in vivo DTI sensitivity and specificity of Ќ to demyelination and remyelination along with ʈ to axonal damage associated with cupri...