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Kevin L. Schey, David M. Anderson, Daniel Mills, Jeffrey Spraggins, Wendi S. Lambert, David J. Calkins; High Resolution MALDI Imaging Methods for Measuring Protein and Lipid Distributions in Rodent Optic Nerves. Invest. Ophthalmol. Vis. Sci. 2011;52(14):6612.
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© ARVO (1962-2015); The Authors (2016-present)
To develop sample preparation and imaging acquisition methods for a novel imaging modality, MALDI mass spectrometry imaging, to determine lipid and protein distributions in rodent optic nerve tissue. The method can then be applied to rodent glaucoma models.
Rodent (C57 mice and Sprague Dawley rat) eyes with optic nerves attached were dissected and placed in ice cold water and rapidly frozen. Twelve micron thick cryosections of the optic nerve tissue were taken both longitudinally and in cross section and placed on a MALDI target. For lipid imaging, sections were washed with ammonium acetate and 2,5-dihydroxybenzoic acid MALDI matrix was sublimated onto the target. For protein imaging, sections were spray coated with sinapinic acid. MALDI spectra were acquired across the sections in 10 um steps (lipids) or 50 um steps (proteins). Images were assembled using Bruker’s FlexImaging software. Lipid identifications were made by accurate mass measurement and tandem mass spectrometry using a 9.4T Bruker Apex-Qe FT-ICR instrument.
Abundant lipid signals were acquired from optic nerve tissue. Images acquired at 10 micron resolution showed several lipid signals localized to peripheral meninges tissue whereas others more centrally localized to axonal-glial tissue. Identification of several sulfatide species is consistent with known nerve lipid composition. Protein profiling experiments revealed signals between m/z 4800 - 18500. Imaging showed a number of these signals to be located within the nerve fiber tissue.
New methodology has been developed to simultaneously image multiple lipids or multiple protein species in rodent optic nerve tissue with high (10-50 micron) spatial resolution. This method is now amenable to imaging lipid and proteins in diseased (glaucomatous) tissue in rodent models.
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