We propose to investigate the distribution of lipids in and near spinal cord tissue using direct examination of untreated tissue sections.
The aim i s to map the distribution of lipids characteristic of the myelin sheath in healthy mice and in animals with neural degenerative disease. It is expected that differences in less polar lipid distributions will occur and might be characteristic of disease state, but the spatial distribution in and near the nerves and the exact nature of the compounds that will be observed is unknown, since previous measurements have either been made with spectroscopic methods with limited molecular recognition capabilities (e.g. CARS) or with severely limited spatial resolution (e.g. extraction followed by LC-ESI-MS).
One aim of the study is to identify lesions through changes in chemical composition and to correlate structural and chemical changes associated with lesions. This will be achieved by a combination of existing optical methods, especially coherent anti-Stokes raman spectroscopy (CARS) with new ambient mass spectrometry methods. The experiments will require the development of a high resolution (ca 10 micron) imaging capability for desorption electrospray ionization mass spectrometry (DESI-MS). DESI-MS is an ambient ionization method that allows rapid and direct chemical analysis of tissue sections with no prior sample treatment. In order to detect lipids with high sensitivity in the tissue matrix, capabilities for reactive DESI will need to be developed;this method involving in situ localized derivatization of the lipid using a charge-labeled reagent which is carried to the spot being interrogated by the spray solvent. Tandem mass spectrometry will be used to further improve the specificity of detection of the lipids of interest. The large amounts of molecular information obtained during MS/MS chemical imaging require comprehensive analysis using advanced data processing methods and new techniques for data representation and visualization, data searching and image comparisons will be implemented. In preliminary work at lower spatial resolution, DESI-MS has been demonstrated to allow differentiation of tumor and non-tumor sections of tissue through spatial distributions of polar lipids, especially phospholipids. Although this technique has many potential applications involving in-vitro and in-vivo chemical analysis for biological systems, in this R21 application, we concentrate on technology development aimed at providing the spatial and chemical information needed t characterize neural degeneration and specifically demyelin.
It is proposed to develop new imaging methods which can be used to identify lipids and other chemicals that might serve as biomarkers to follow the fundamental processes occurring in the course of neural diseases like multiple sclerosis (MS). Specific information on the chemical changes in the nerve sheath will be sought in order to allow future diagnosis and prognosis of neural diseases.
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