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Diffusion basis spectrum imaging detects and distinguishes coexisting subclinical inflammation, demyelination and axonal injury in experimental autoimmune encephalomyelitis mice.

ABSTRACT: Clinicopathological paradox has hampered significantly the effective assessment of the efficacy of therapeutic intervention for multiple sclerosis. Neuroimaging biomarkers of tissue injury could guide more effective treatment by accurately reflecting the underlying subclinical pathologies. Diffusion tensor imaging-derived directional diffusivity and anisotropy indices have been applied to characterize white matter disorders. However, these biomarkers are sometimes confounded by the complex pathologies seen in multiple sclerosis and its animal models. Recently, a novel technique of diffusion basis spectrum imaging has been developed to quantitatively assess axonal injury, demyelination and inflammation in a mouse model of inflammatory demyelination. Lenaldekar, which inhibits T-cell expansion in a non-cytolytic manner, has been shown to suppress relapses and preserve white matter integrity in mice with experimental autoimmune encephalomyelitis. In this study, relapsing-remitting experimental autoimmune encephalomyelitis was induced through active immunization of SJL/J mice with a myelin proteolipid protein peptide. The therapeutic efficacy of Lenaldekar treatment was evaluated via daily clinical score, cross-sectional ex vivo diffusion basis spectrum imaging examination and histological analysis. Lenaldekar greatly reduced relapse severity and protected white matter integrity in these experimental autoimmune encephalomyelitis mice. Diffusion basis spectrum imaging-derived axial diffusivity, radial diffusivity and restricted diffusion tensor fraction accurately reflected axonal injury, myelin integrity and inflammation-associated cellularity change, respectively. These results support the potential use of diffusion basis spectrum imaging as an effective outcome measure for preclinical drug evaluation.

SUBMITTER: Wang X

PROVIDER: S-EPMC4071074 | biostudies-other | 2014 Jul

REPOSITORIES: biostudies-other

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Project description:BackgroundMultiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS) characterized by inflammation, demyelination and axonal pathology. Myelin basic protein/proteolipid protein (MBP-PLP) fusion protein MP4 is capable of inducing chronic experimental autoimmune encephalomyelitis (EAE) in susceptible mouse strains mirroring diverse histopathological and immunological hallmarks of MS. Lack of human tissue underscores the importance of animal models to study the pathology of MS.MethodsTwenty-two female C57BL/6 (B6) mice were immunized with MP4 and the clinical development of experimental autoimmune encephalomyelitis (EAE) was observed. Methylene blue-stained semi-thin and ultra-thin sections of the lumbar spinal cord were assessed at the peak of acute EAE, three months (chronic EAE) and six months after onset of EAE (long-term EAE). The extent of lesional area and inflammation were analyzed in semi-thin sections on a light microscopic level. The magnitude of demyelination and axonal damage were determined using electron microscopy. Emphasis was put on the ventrolateral tract (VLT) of the spinal cord.ResultsB6 mice demonstrated increasing demyelination and severe axonal pathology in the course of MP4-induced EAE. Additionally, mitochondrial swelling and a decrease in the nearest neighbor neurofilament distance (NNND) as early signs of axonal damage were evident with the onset of EAE. In semi-thin sections we observed the maximum of lesional area in the chronic state of EAE while inflammation was found to a similar extent in acute and chronic EAE. In contrast to the well-established myelin oligodendrocyte glycoprotein (MOG) model, disease stages of MP4-induced EAE could not be distinguished by assessing the extent of parenchymal edema or the grade of inflammation.ConclusionsOur results complement our previous ultrastructural studies of B6 EAE models and suggest that B6 mice immunized with different antigens constitute useful instruments to study the diverse histopathological aspects of MS.

Project description:We previously identified that ngr1 allele deletion limits the severity of experimental autoimmune encephalomyelitis (EAE) by preserving axonal integrity. However, whether this favorable outcome observed in EAE is a consequence of an abrogated neuronal-specific pathophysiological mechanism, is yet to be defined. Here we show that, Cre-loxP-mediated neuron-specific deletion of ngr1 preserved axonal integrity, whereas its re-expression in ngr1-/- female mice potentiated EAE-axonopathy. As a corollary, myelin integrity was preserved under Cre deletion in ngr1flx/flx , retinal ganglion cell axons whereas, significant demyelination occurred in the ngr1-/- optic nerves following the re-introduction of NgR1. Moreover, Cre-loxP-mediated axon-specific deletion of ngr1 in ngr1flx/flx mice also demonstrated efficient anterograde transport of fluorescently-labeled ChTxβ in the optic nerves of EAE-induced mice. However, the anterograde transport of ChTxβ displayed accumulation in optic nerve degenerative axons of EAE-induced ngr1-/- mice, when NgR1 was reintroduced but was shown to be transported efficiently in the contralateral non- recombinant adeno-associated virus serotype 2-transduced optic nerves of these mutant mice. We further identified that the interaction between the axonal motor protein, Kinesin-1 and collapsin response mediator protein 2 (CRMP2) was unchanged upon Cre deletion of ngr1 Whereas, this Kinesin-1/CRMP2 association was reduced when NgR1 was re-expressed in the ngr1-/- optic nerves. Our data suggest that NgR1 governs axonal degeneration in the context of inflammatory-mediated demyelination through the phosphorylation of CRMP2 by stalling axonal vesicular transport. Moreover, axon-specific deletion of ngr1 preserves axonal transport mechanisms, blunting the induction of inflammatory demyelination and limiting the severity of EAE.SIGNIFICANCE STATEMENT Multiple sclerosis (MS) is commonly induced by aberrant immune-mediated destruction of the protective sheath of nerve fibers (known as myelin). However, it has been shown that MS lesions do not only consist of this disease pattern, exhibiting heterogeneity with continual destruction of axons. Here we investigate how neuronal NgR1 can drive inflammatory-mediated axonal degeneration and demyelination within the optic nerve by analyzing its downstream signaling events that govern axonal vesicular transport. We identify that abrogating the NgR1/pCRMP2 signaling cascade can maintain Kinesin-1-dependent anterograde axonal transport to limit inflammatory-mediated axonopathy and demyelination. The ability to differentiate between primary and secondary mechanisms of axonal degeneration may uncover therapeutic strategies to limit axonal damage and progressive MS.

Project description:Globoid cell leukodystrophy is an inherited neurodegenerative disorder caused by a deficiency of the lysosomal enzyme galactosylceramidase. In both human patients and the authentic murine Twitcher model, pathological findings include demyelination as well as axonal damage in both the central and peripheral nervous system. Diffusion tensor imaging (DTI) has emerged as a powerful noninvasive technique that is sensitive to these white matter disease processes. Increases in radial diffusivity (lambda perpendicular) and decreases in axial diffusivity (lambda parallel) correlate with histopathological evidence of demyelination and axonal damage, respectively. Compared to age-matched, normal littermates, DTI of optic nerve and trigeminal nerve in end-stage Twitcher mice displayed a statistically significant increase in lambda perpendicular and decrease in lambda parallel, consistent with previously characterized demyelination and axonal damage in these regions. In the Twitcher spinal cord, a statistically significant decrease in lambda parallel was identified in both the dorsal and ventrolateral white matter, relative to normal controls. These results were consistent with immunofluorescence evidence of axonal damage in these areas as detected by staining for nonphosphorylated neurofilaments (SMI32). Increase in lambda perpendicular in Twitcher spinal cord white matter relative to normal controls reached statistical significance in the dorsal columns and approached statistical significance in the ventrolateral region. Correlative reduced levels of myelin basic protein were detected by immunofluorescent staining in both these white matter regions in the Twitcher spinal cord. Fractional anisotropy, a nonspecific but sensitive indicator of white matter disease, was significantly reduced in the optic nerve, trigeminal nerve, and throughout the spinal cord white matter of Twitcher mice, relative to normal controls. This first reported application of spinal cord DTI in the setting of GLD holds potential as a noninvasive, quantitative assay of therapeutic efficacy in future treatment studies.

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Diffusion basis spectrum imaging detects and distinguishes coexisting subclinical inflammation, demyelination and axonal injury in experimental autoimmune encephalomyelitis mice.

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