Investigating biochemical and structural changes in animal models of multiple sclerosis using Fourier transform infrared imaging and small angle x-ray scattering
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thesis
posted on 2017-05-15, 07:06authored byCaine, 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.
History
Campus location
Australia
Principal supervisor
Donald McNaughton
Additional supervisor 1
Claude Bernard, Philip Heraud, Mark Tobin
Year of Award
2012
Department, School or Centre
Chemistry
Additional Institution or Organisation
Centre for Biospectroscopy. Monash Immunology and Stem Cell Laboratories