Mass spectrometry imaging of biomarker lipids for phagocytosis and signalling during focal cerebral ischaemia

Focal cerebral ischaemia has an initial phase of inflammation and tissue injury followed by a later phase of resolution and repair. Mass spectrometry imaging (desorption electrospray ionization and matrix assisted laser desorption ionization) was applied on brain sections from mice 2 h, 24 h, 5d, 7d, and 20d after permanent focal cerebral ischaemia. Within 24 h, N-acyl-phosphatidylethanolamines, lysophosphatidylcholine, and ceramide accumulated, while sphingomyelin disappeared. At the later resolution stages, bis(monoacylglycero)phosphate (BMP(22:6/22:6)), 2-arachidonoyl-glycerol, ceramide-phosphate, sphingosine-1-phosphate, lysophosphatidylserine, and cholesteryl ester appeared. At day 5 to 7, dihydroxy derivates of docosahexaenoic and docosapentaenoic acid, some of which may be pro-resolving mediators, e.g. resolvins, were found in the injured area, and BMP(22:6/22:6) co-localized with the macrophage biomarker CD11b, and probably with cholesteryl ester. Mass spectrometry imaging can visualize spatiotemporal changes in the lipidome during the progression and resolution of focal cerebral inflammation and suggests that BMP(22:6/22:6) and N-acyl-phosphatidylethanolamines can be used as biomarkers for phagocytizing macrophages/microglia cells and dead neurones, respectively.

3 adduct ion and its MS/MS spectrum at m/z 769. 5. The corresponding molecular structures to the assigned letters in the spectra can be seen in Supplementary Table S1. adduct. For (c) and (d) it was not possible to identify the molecular structure by MS/MS, however, we still believe that this in fact is Cer(d18:1/18:0) in accordance with results of others 1 . The corresponding molecular structures to the assigned letters in the spectra can be seen in Supplementary Table S1. Figure S4. PC(10:0/10:0) sprayed on section as internal standard: To test whether the ion suppression in the ischaemic area differed from the healthy brain tissue, we sprayed a section with 24h post-surgical survival with PC(10:0/10:0) (not naturally present in the brain). Especially the sodium adduct 5 but also to a lesser extent the potassium adduct for PC(10:0/10:0) seemed to accumulate more easily in the ischaemic area. However, this apparent smaller ion suppression in the ischaemic area appear not sufficient to explain the accumulation of e.g. LysoPC(16:0) and Cer(d18:1/18:0) during ischaemia. The ion images have individual intensity bars between 0-100%, and therefore, the intensity colours cannot be compared between two images. The images were measured in positive ion mode by DESI imaging with a spatial resolution of 120x120 µm 2 and the images are a typical representative of 2 mice. High magnification photomicrographs of a brain section from a mouse subjected to pMCAO with 5 days post-surgical survival and stained for CD11b.

Supplementary Figure S14. Accumulation of BMP and LysoPS in TiO 2 -nanoparticle-exoposed mouse
lungs: BMP is especially abundant in alveolar macrophages and we, therefore, analysed the distribution of BMP species in control mouse lungs and TiO 2 -nanoparticle-exposed mouse lungs. This showed that BMP(36:2) and BMP(22:6/22:6) were increased in the TiO 2 -nanoparticle-exposed lungs compared to the controls. Furthermore, LysoPS(18:0) had also increased in the TiO 2 -nanoparticle-exposed lungs. PG(34:1) also seemed to be increased in the TiO 2 -nanoparticle-exposed lungs compared to the control, however, not to the same extent. The ion images have individual intensity bars between 0-100%, and therefore, the intensity colours cannot be compared between two images. The images were measured in negative ion mode by MALDI imaging with a spatial resolution of 50x50 µm 2 Supplementary Table S1 3 were carried out on mouse brains to identify the molecular structures of lipid species using DESI on a LTQ XL linear ion trap mass spectrometer (Thermo Scientific, USA) equipped with a custom-built DESI imaging ion source, as previously described 6 .

MS/MS and MS 3 measurements on mouse brains. MS/MS and MS
An m/z value of interest was separated and fragmented by Collision-Induced Dissociation (CID) (MS/MS).
Using the m/z values of the fragments, the molecular structure could be identified. In some cases the most abundant fragment was separated and then fragmented by CID giving further molecular structure information (MS 3 ). For some lipids the spatial localization of their CID fragments were investigated by DESI imaging, which was carried out as described in the method section.
Spraying section with lipid standard. A Brain section with 24h post-surgical survival was placed on a moving stage and with a spray pointed directly towards the section with a spray-to-sample distance of 3 cm.
The spray was constructed of coaxial fused silica capillaries connected in 1/16-inch Swagelok tee (Swagelok Co., USA), an inner capillary (50 µm ID, 150 µm OD, SGE, USA) carrying the spray solvent, and an outer capillary (250 µm ID, 350 µm OD, SGE, USA) carrying the nebulizer gas. The spray consisted of 0.01 mg/mL PC(10:0/10:0) in methanol and water (95:5) dispensed with a flow of 10 µl/min and a nitrogen nebulizer gas pressure of 2 bar. The section was moved under the spray with a speed of 10 mm/s in a meandering pattern across the section and this was repeated over 10 cycles. Afterwards the Section was measured by DESI imaging as described in the method section.
Cresyl violet acetate stains. Sections were after end of the measurement stained with 0.5% Cresyl violet acetate (Sigma-Aldrich, USA) in water for approximately 8 min. and then dehydrated in graded series of alcohol (70-99%), cleared in xylene, and finally cover-slipped with Eukitt quick-hardening mounting medium (Fluka Analytical, Sigma-Aldrich, Missouri, USA).The Cresyl violet acetate (CV) stains were then captured on a Stemi DV4 Stereoscope (Carl Zeiss AG, Oberkuchen, Germany) equipped with an LCMOS digital streaming camera (Brunel Microscopes Ltd, Chippenham, UK). Images composed of more than one image were stitched by the Image Composite Editor (Microsoft Corporation, Washington, USA).