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David M. Anderson, Jeffrey Spraggins, Max Joffee, Wendi S. Lambert, Kevin L. Schey, David J. Calkins; Imaging Mass Spectrometry of Glaucoma Model Rodent Retina and Optic Nerve. Invest. Ophthalmol. Vis. Sci. 2012;53(14):2479.
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© ARVO (1962-2015); The Authors (2016-present)
Glaucoma blinds in a sectorial pattern of retinal and optic nerve degeneration. Matrix Assisted Laser Desorption Ionization Imaging Mass Spectrometry (MALDI-IMS) is being used increasingly to determine the spatial distribution of proteins, lipids and small molecules in a variety of tissues. Here we used MALDI-IMS to compare distributions of lipids and energy metabolites in retinas and optic nerves of C57 mice with microbead-induced elevations in ocular pressure and DBA/2J mice.
Using C57 mice for lipid imaging, we elevated ocular pressure in the left eye 25-30% using microbead occlusion of the anterior chamber for 4/6 months; the right eye received an equivalent volume saline injection as a control. Whole eyes including optic nerve were removed fresh and rapidly frozen in deionized water. We prepared 12 µm sections with a cryostat and thaw-mounted onto a gold coated plate. The sections were desiccated and washed with 100mM ammonium acetate before MALDI matrix, dihydroxybenzoic acid, was applied via sublimation. Optic nerve tissue was imaged using a Bruker UltrafleXtreme II at a spatial resolution of 10 µm. Lipid identification was performed using a 9.4T Bruker Apex Qe FT-ICR mass spectrometer. Images were generated using Bruker FlexImaging software. Retinas for metabolite imaging were removed from 3- and 9- month DBA/2J mice and flat-mounted between two cover slips. Samples were snap frozen, lyophilized, and attached sclera side down onto double sided carbon tape. 9-Aminoacridine matrix was applied via air spray deposition and images were acquired in MS/MS mode using a Thermo LTQ instrument equipped with a MALDI source at 100 µm spatial resolution.
MALDI images were produced from a number of phosphatidylcholine and sulfatide lipid species in mouse disease model and control optic nerve tissue. Several lipid species such as PC (18:1/16:0) exhibit altered relative abundances between the control and disease models sections where differences appeared in sectorial fashion. Differences in relative abundance have also being observed along the nerve fiber (anterior vs. posterior). Metabolite images from DBA/2J retina showed the relative abundance of ADP increased at 9 months with a concordant decrease in the abundance of ATP.
MALDI imaging methods have been used to identify sectorial differences and spatial localization of lipid species and an energy metabolite in elevated ocular pressure mouse model tissues.
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