Visualising Cholesterol in Brain by On-Tissue Derivatisation and Quantitative Mass Spectrometry Imaging

Abstract

Despite being a critical molecule for neurobiology and brain health, mass spectrometry imaging (MSI) of cholesterol has been under reported compared to other lipids, due to the difficulty in ionising the sterol molecule. In the present work we have employed an on-tissue enzyme-assisted derivatisation strategy to improve detection of cholesterol in brain tissue sections. We report distribution and levels of cholesterol across specific brain structures of the mouse brain, in a model of Niemann-Pick type C1 (NPC1) disease, and during brain development. MSI revealed how cholesterol changes during development and that in the adult is highest in pons and medulla of the brain stem. Cholesterol was significantly reduced in the corpus callosum and other brain regions in the Npc1 null mouse, confirming hypomyelination at the molecular level. Our study demonstrates the potential of MSI to the study of sterols in neuroscience.

Mass Spectrometry Imaging of Cholesterol and Oxysterols

“…Cholesterol and most oxysterols, however, do not possess an oxo group and are not suitable for direct derivatisation with hydrazine reagents. This obstacle is overcome for in-solution derivatisation by incorporating an enzymatic oxidation step prior to treatment with Girard reagent (41, 42), and can be similarly adopted as part of the on-tissue derivatisation approach (6,43). This on-tissue "enzymeassisted derivatisation for sterol analysis" (EADSA) approach has been successfully adopted for imaging cholesterol in rodent and human brain, liver tissue and in the developing zebrafish.…”

“…Bacterial cholesterol oxidase is next sprayed onto the tissue in phosphate buffer, the tissue is then incubated for 1 hr in a humid atmosphere. Cholesterol oxidase enzyme oxidises 3β-hydroxy-5-ene sterols to their 3-oxo-4-ene analogues (Figure 4) (44), and after drying the tissue again in a desiccator it is sprayed with GP reagent in aqueous acidic methanol, then incubated in an atmosphere of aqueous acidic methanol (6,43). The tissue is dried once more.…”

“…At its simplest, a probe (e.g., laser or ion beam) samples the surface of a tissue section pixel by pixel with mass spectra being recorded of the sampled-surface at each pixel. An image of any ion recorded by the MS can then be generated across the sampled surface with ion abundance pixel by pixel illustrated on a colour scale (Figure 1) (5,6). As an ion's mass/charge (m/z), as recorded by MS, is related to its parent compound's molecular mass, and hence to a particular compound, MSI at least to a first approximation, gives a molecular image of a compound in a tissue.…”

“…As an ion's mass/charge (m/z), as recorded by MS, is related to its parent compound's molecular mass, and hence to a particular compound, MSI at least to a first approximation, gives a molecular image of a compound in a tissue. There are many differing sampling probes providing different degrees of spatial resolution, ranging from 100 to 400 nm or smaller for secondary ion mass spectrometry (SIMS) at the high spatial resolution end of the spectrum (7,8); 50 to 100 µm typical for matrix-assisted laser desorption ionisation (MALDI) (4)(5)(6) and desorption electrospray ionisation ionisation (DESI) (9) and 20 µm or less for the more sensitive MALDI2 (10,11); to 300 -1,000 µm for liquid extraction for surface analysis (LESA) at the low spatial resolution end of the spectrum (12,13). However, as spatial resolution is increased the pixel size get smaller and the available tissue to be analysed is reduced meaning that low-abundance metabolites are discriminated against at high spatial resolution, this is less of a problem for highly abundant cholesterol but is for its lower abundance precursors and metabolites.…”

Aziridination-Assisted Mass Spectrometry of Nonpolar Sterol Lipids with Isomeric Resolution

“…Atmospheric-pressure chemical ionization (APCI), 21 matrixassisted laser dissociation ionization (MALDI), 22 and direct analysis in real time (DART) 23 have all been used to facilitate ionization for the analysis of nonpolar lipids with more effective ionization efficiencies than those seen with ESI. 24 Likewise, derivatization methods targeting hydroxy 25,26 and carboxyl 27 groups were shown to facilitate the visualization of nonpolar lipids in MS. However, these methods do not provide C�C bond isomer resolution.…”