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Investigating biochemical and structural changes in animal models of multiple sclerosis using Fourier transform infrared imaging and small angle x-ray scattering
thesisposted on 15.05.2017, 07:06 by Caine, Sally
Multiple sclerosis (MS) is a debilitating disease of the central nervous system and is the leading cause of non-traumatic neurological disability in young adults. MS affects over 2 million people worldwide and is commonly believed to arise from an autoimmune attack directed against components of the myelin sheath, resulting in multifocal lesions characterised by inflammation, demyelination and axonal damage. A complex interplay between genetic and environmental factors is thought to contribute to disease susceptibility. Although conventional histopathological assessment and magnetic resonance imaging techniques have greatly improved our understanding of lesion activity, the aetiology of MS and the mechanisms underlying lesion formation remain largely unknown. In recent decades, advances in instrumentation and multivariate analysis tools have seen Fourier transform infrared (FTIR) microspectroscopic imaging become a powerful tool for detecting discrete and subtle changes in the macromolecular composition of healthy and diseased tissues. Complementary information about the fundamental structural attributes of the myelin sheath and quantification of the relative amount of myelin within the sample can be determined using small angle X-ray scattering (SAXS). The aim of this thesis was to investigate the biochemical and structural changes underpinning the pathological, developmental and reparative processes in animal models of MS using FTIR microspectroscopic imaging and SAXS. In the first part of this thesis, laboratory based FTIR microspectroscopic imaging, bioinformatics, and synchrotron mapping were used to analyse macromolecular changes in the CNS during the course of experimental autoimmune encephalomyelitis (EAE), an animal model of MS. Using this approach, the distinct and heterogeneous tissue layers of the cerebellum and spinal cord, as well as lesion pathology, could be distinguished from one another. EAE lesions were characterised by low relative lipid concentrations and high relative nucleic acid concentrations and correlated well with regions of demyelination and inflammation identified using conventional histological and immunofluorescence staining techniques. The identification unique infrared ‘spectral phenotypes' identified allowed the training of artificial neural networks (ANNs) capable of discriminating EAE pathology from the surrounding healthy tissue, in an unbiased and automated fashion. Moreover, the integration of ANNs with the higher lateral resolution achieved using synchrotron mapping allowed the early detection and definitive identification of microlesions in the CNS of mice, prior to the onset of clinical signs of EAE. Furthermore, the potential of this technique for the evaluation of new therapeutic agents was demonstrated in lesions of animals partially protected against EAE by vaccination with Nogo-A, an inhibitor of neurite outgrowth, where subtle chemical and protein secondary structural changes, not observed by conventional histology, were identified. For the second part of this thesis, FTIR microspectroscopic imaging and SAXS, in conjunction with conventional histological and EM techniques were used to detect and characterise the natural biochemical and ultrastructural changes associated with developmental myelination in the corpus callosum of healthy mice. The onset of myelination was consistently found to occur at postnatal day 14 (P14), while the rate of myelination varied depending on the analytical employed. Myelination reached a maximum rate between P14‒P21 and P21‒P28 as determined by SAXS and EM, respectively. In contrast, the rate of myelination was found to increase at a constant rate when measured by the relative amount of lipid quantified by FTIR microspectroscopic imaging. In addition to biochemical changes, SAXS analysis revealed that the myelin sheath underwent significant compaction at the extracellular space, which coincided with alterations in protein secondary structure detected in the FTIR spectra. Together, these data suggest that proteins involved in the compaction of the myelin sheath at this site, are responsible for the observed FTIR spectroscopic changes. The identification of significant biochemical changes between the oldest animals (P140) used in this study and the mice aged P98 and younger is of considerable importance, as mice aged between P56‒P84 are often used in research and thus may still be undergoing significant developmental changes. This is particularly relevant to the CPZ intoxication animal model, which is increasingly being used in MS research to assess demyelination and remyelination of the corpus callosum of mice aged between P56 and P140. The data obtained here, therefore provide a useful benchmark against which the biochemical and ultrastructural changes occurring in the corpus callosum following CPZ intoxication can be identified and compared. The ability of FTIR microspectroscopic imaging and SAXS to detect and quantify relative biochemical and structural changes during chemically induced demyelination and following subsequent remyelination of the corpus callosum in the CPZ intoxication animal model was examined in the third part of this thesis. Changes in the relative amounts of demyelination and remyelination were easily visualised and quantified in the FTIR spectra using the integrated area of the lipid ester carbonyl band as a measure of myelin. Notably, alterations in protein secondary structure were identified following remyelination, suggesting that such differences could be used to identify remyelination in a rapid and automated fashion. Despite these protein conformational changes, the ultrastructure of the myelin sheath, including the widths of the myelin period, lipid bilayers, cytoplasmic space and extracellular space, did not significantly differ during demyelination or remyelination, when compared with the age-matched controls. Interestingly, a discrepancy between the relative amount of myelin measured by SAXS and the average number of myelinated axons within the electron micrographs was found, suggesting that the SAXS technique is only capable of detecting myelin in a highly ordered structure. Thus, the SAXS method applied here could serve as a rapid means for quantifying the relative amount of intact internodal myelin within a sample and could be used to assess the effect of novel therapies on the relative amount of myelin remaining after demyelination or accumulating following remyelination. In summary, the data presented in this thesis illustrates the power of FTIR microspectroscopic imaging and SAXS techniques to detect subtle biochemical and structural changes associated with CNS pathology. These two techniques form a powerful addition to conventional techniques, providing rich biochemical and structural information and a unique opportunity to investigate a range of CNS pathologies within tissues at the molecular level, as well as the potential to evaluate and understand new therapeutic approaches and mechanisms of action.