Distinguishing core from penumbra by lipid profiles using Mass Spectrometry Imaging in a transgenic mouse model of ischemic stroke

Abstract

Detecting different lipid profiles in early infarct development may give an insight on the fate of compromised tissue. Here we used Mass Spectrometry Imaging to identify lipids at 4, 8 and 24 hours after ischemic stroke in mice, induced by transient middle cerebral artery occlusion (tMCAO). Combining linear transparency overlay, a clustering pipeline and spatial segmentation, we identified three regions: infarct core, penumbra (i.e. comprised tissue that is not yet converted to core), and surrounding healthy tissue. Phosphatidylinositol 4-phosphate (m/z = 965.5) became visible in the penumbra 24 hours after tMCAO. Infarct evolution was shown by 2D-renderings of multiple phosphatidylcholine (PC) and Lyso-PC isoforms. High-resolution Secondary Ion Mass Spectrometry, to evaluate sodium/potassium ratios, revealed a significant increase in sodium and a decrease in potassium species in the ischemic area (core and penumbra) compared to healthy tissue at 24 hours after tMCAO. In a transgenic mouse model with an enhanced susceptibility to ischemic stroke, we found a more pronounced discrimination in sodium/potassium ratios between penumbra and healthy regions. Insight in changes in lipid profiles in the first hours of stroke may guide the development of new prognostic biomarkers and novel therapeutic targets to minimize infarct progression.

Figure 1

A schematic representation of the manual overlays of MALDI-MSI and Nissl stain with marked necrotic border in SCiLS Lab 2016b. The manual overlay was followed by a linear transparency overlay (a) of a selected m/z value (in the example m/z = 782.6 ± 0.3). The high intensity of the [PC(34:1) + Na]⁺ perfectly aligns within the necrotic border of the Nissl stain. (b) In the next step, the manual overlay was followed by a spatial segmentation pipeline built in the software. Once the segmentation map was created it detected prominent spatial regions ipsilateral, representing core, penumbra and healthy tissue, here at 4 hours after transient middle cerebral artery occlusion.

Figure 2

MALDI images showing the distribution of the PIP molecular family and accompanying Nissl stainings. Selected MALDI m/z images acquired at 50-µm spatial resolution showing the distribution of specific lipids m/z = 885.6 [PI(38:4) − H]⁻, m/z = 965.5 [PIP(38:4) − H]⁻, and m/z = 1045.5 [PIP₂(38:4) − H]⁻ in a SHAM WT mouse and at 4, 8 and 24 hours after transient middle cerebral artery occlusion measured in negative ion mode. The last column represents the linear transparency overlays of m/z = 885.6 [PI(38:4) − H]⁻, m/z = 965.5 [PIP(38:4) − H]⁻, and m/z = 1045.5 [PIP₂(38:4) − H]⁻ with Nissl staining at 24 hours after tMCAO.

Figure 3

Probabilistic latent semantic analysis (pLSA) loading plots and spectra of resulting components of the WT mice in negative (components 4 and 9) and positive (components 1, 2 and 7) ion mode 24 hours after transient middle cerebral artery occlusion.

Figure 4

MALDI images showing the distribution of lyso-lipids and accompanying Nissl stainings. Selected MALDI m/z images acquired at 50-µm spatial resolution showing the distribution of lyso-lipids in a SHAM WT mouse and at 4, 8 and 24 hours after transient middle cerebral artery occlusion measured in positive ion mode. Each row of the selected species represents un-washed (top) and washed (bottom) tissue section.

Figure 5

MRI, SIMS and MALDI images revealing the distribution of the infarct core in wild-type mice after transient middle cerebral artery occlusion. (a) PC4 loadings plot revealing the distribution of co-related species with Na⁺ (positive loadings plot-green) and K⁺ (negative loadings-blue), (b) RGB overlay of three PCs (red; PC-3, green; PC4, and blue; PC-4); (c) T2-weighted MRI image with delineated infarct border; MALDI image of (d) m/z = 756.6 [PC(32:0) + Na]⁺ and (e) m/z = 772.6 [PC(32:0) + K]⁺; (f) K⁺/Na⁺ ratio, (g) choline fragment, (h) phosphatidylcholine fragment in positive ion mode and (i) palmitic acid (16:0) m/z = 255, in ischemia 24 hours after transient middle middle cerebral artery occlusion.

Figure 6

Linear discriminant analysis histograms of wild-type (top row) and transgenic FHM1 mice (bottom row) of segmented ROI areas (healthy (black), ischemic (light grey) and borderzone (dark grey)) at 4, 8 and 24 hours after transient middle cerebral artery occlusion.