Date of Award

2013

Degree Type

Thesis

Degree Name

Doctor of Philosophy

School

School of Medical Science

Faculty

Computing, Health and Science

First Advisor

Dr Wayne Leifert

Second Advisor

Prof Ralph Martins

Third Advisor

Prof Michael Fenech

Abstract

Previous studies have shown that mild cognitive impairment (MCI) may reflect the early stages of more pronounced neurodegenerative disorders such as Alzheimer’s disease (AD). In clinical practice, patients with AD are not usually identified until the disease has progressed to a stage when primary prevention is no longer possible. Therefore there is a need for a minimally invasive and inexpensive diagnostic to identify those who exhibit cellular pathology indicative of MCI and AD risk so that they can be prioritised for primary prevention. Human buccal cells are accessible in a minimally invasive manner, and exhibit cytological and nuclear morphologies that may be indicative of accelerated ageing or neurodegenerative disorders such as AD. The hypothesis that a minimally invasive approach using isolated buccal mucosa cells can be used to identify individuals diagnosed with MCI or AD was therefore tested using laser scanning cytometry (LSC). LSC combines the principles of flow cytometry, quantitative imaging and immunohistochemistry with high-content, multi-color fluorescence analysis, and can be used to identify specific cells in a heterogeneous population as well as scoring unique molecular events within them. This study aimed at investigating buccal cell types (buccal cell cytome) by the use of high-content LSC analysis and to detect potential biomarkers of MCI and AD risk i.e. buccal cell types, nuclear DNA content, intracellular neutral lipids, Tau protein and amyloid-β (Aβ) protein. Buccal cells were sampled from the South Australian Alzheimer’s Nutrition & DNA Damage study (SAND) or the The Australian Imaging, Biomarker & Lifestyle Flagship Study of Ageing (AIBL), fixed and stained with labelled fluorescent antibodies (for detection of Aβ and Tau) and/or DAPI, Fast Green and Oil Red O dyes. In an initial study an LSC protocol was developed to identify and measure differences in buccal cell types and nuclear DNA content as well as a significant increase in micronuclei measured in AD (n=10) and Down’s syndrome (n=10) compared to their respective controls (n=20). Another LSC protocol measured a significant increase in DNA content and hyperdiploidy (as measured by DAPI fluorescence) as well as a significant decrease in neutral lipid content (measured by Oil Red O staining) in buccal cells of MCI (n=22) and AD (n=15) compared to controls (n=37) from the SAND study. Using another novel LSC protocol a significant increase in Aβ was measured in buccal cells from AD (n=20) compared to controls (n=20) from the AIBL study. Immunocytochemistry and ELISA experiments showed no significant differences in putative buccal cell Tau protein. The diagnostic value of parameters examined in these studies, individually or in combination was assessed and reported as specificity and sensitivity scores. In these studies, LSC has proven to be an efficient and useful technology for high-content analysis of buccal cells. Moreover, the changes in the buccal cell cytome observed using LSC may reflect alterations in the metabolism, cellular kinetics, gene expression, genome stability or structural profile of the buccal mucosa, and may prove useful as potential biomarkers in identifying individuals with a high risk of developing MCI and eventually AD.

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