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, 67, 51-63

Imaging Mass Spectrometry of Frontal White Matter Lipid Changes in Human Alcoholics

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Imaging Mass Spectrometry of Frontal White Matter Lipid Changes in Human Alcoholics

Suzanne M de la Monte et al. Alcohol.

Abstract

Background: Chronic alcohol use disorders (AUD) are associated with white matter (WM) degeneration with altered myelin integrity. Matrix assisted laser desorption ionization-imaging mass spectrometry (MALDI-IMS) enables high throughput analysis of myelin lipid biochemical histopathology to help characterize disease mechanisms.

Purpose: This study utilized MALDI-IMS to investigate frontal lobe WM myelin lipid abnormalities in AUD.

Methods: Standardized cores of formalin-fixed WM from Brodmann Area 4 (BA4) and BA8/9 of 20 postmortem AUD and 19 control adult human brains were embedded in carboxymethyl-cellulose, cryo-sectioned (8 μm), thaw-mounted onto indium tin oxide (ITO) -coated glass slides, and sublimed with 2,5-dihydroxybenzxoic acid (DHB) matrix. Lipids were imaged by MALDI-time of flight in the negative ionization mode. Data were visualized with FlexImaging software v4.0 and analyzed with ClinProTools v3.0.

Results: Principal component analysis (PCA) and data bar plots of MALDI-IMS data differentiated AUD from control WM. The dominant effect of AUD was to broadly reduce expression of sphingolipids (sulfatides and ceramides) and phospholipids. Data bar plots demonstrated overall similar responses to AUD in BA4 and BA8/9. However, differential regional effects of AUD on WM lipid profiles were manifested by non-overlapping expression or discordant responses to AUD for a subset of lipid ions.

Conclusions: Human AUD is associated with substantial inhibition of frontal lobe WM lipid expression with regional variability in these effects. MALDI-IMS can be used to characterize the nature of AUD-associated lipid biochemical abnormalities for correlation with lifetime exposures and WM degeneration, altered gene expression, and responses to abstinence or treatment.

Keywords: Alcohol-use disorder; Human; Imaging; Mass spectrometry; Neurodegeneration; Phospholipids; Sphingolipids; White matter.

Figures

Figure 1
Figure 1
Principal component analysis (PCA) of imaging mass spectrometry (IMS) data acquired in the negative ionization mode of MALDI mass spectrometry: ClinProTools was used for PCA of the total IMS spectra generated from (A-D) BA4 and (E-H) BA8/9 control (blue) and AUD (red) frontal lobe white matter (WM) regions of interest (ROIs). Panels A and E represent the 3-D PCA images. B-D and F-H correspond to 2-D rotated images of A and E, respectively. In each region, two distinct but overlapping clusters were identified corresponding to control and AUD samples. Non-overlapping components of each spectra reflect differential effects of AUD on frontal WM lipid expression in BA4 and BA8/9.
Figure 2
Figure 2
Data Bar Plots illustrating effects of AUD on lipid ion expression in BA4 and BA8/9 human frontal lobe WM as detected by MALDI-IMS in the negative ionization mode: Plots depict the calculated percentage differences in the mean levels of each lipid ion expressed. The scale bars depict the range of responses from -80% to +160% relative to control. T-test results (P-Values) from comparing the mean levels of each lipid ion in control and AUD groups are color-coded to the right of each data bar plot. Results are organized with respect to increasing m/z values. See Supplementary Table 1 for lipid identifications. Blue bars to the left of the vertical axis reflect AUD-associated reductions in lipid expression, and red bars to the right correspond to mean percentage increases in lipid expression.
Figure 3
Figure 3
AUD-associated changes (increases or decreases) in the expression of different lipid subtypes: Databar results shown in Figure 2 were sorted and culled to graphically summarize effects of AUD on different lipid subtypes in BA4 (A, C, E) and BA8/9 (B, D, F) of human frontal lobe WM. The graphs depict the percentages of each lipid subtype that were significantly altered in expression level relative to control (increases and decreases were combined). Results were analyzed using Chi-square tests to determine if AUD differentially altered expression of (A, B) sphingolipids versus phospholipids, (C, D) sulfatides versus ceramides, and (E, F) different phospholipid subtypes (PA = phosphatidic acid; PE = phosphatidylethanolamine; PG = phosphatidylglycerol; PI = phosphatidylinositol; PS = phosphatidylserine); P-values are shown within each panel. Phosphatidylcholine (PC) was not detected in the negative ionization mode of mass spectrometry.
Figure 4
Figure 4
Inhibitory versus stimulatory effects of AUD on relative lipid ion expression in the (A, C, E) BA4 and (B, D, F) BA8/9 regions of human frontal lobe WM. Results in Figure 2 are summarized and graphically displayed to compare the percentages of (A, B) sphingolipids and phospholipids, (C, D) sulfatides and ceramides, and (E, F) different phospholipid subtypes that were significantly increased, unchanged, or significantly decreased in AUD relative to control samples. Data were analyzed using Chi-square tests and corresponding P-values are shown within the panels. Phosphatidylcholine was not detected in the negative ionization mode of mass spectrometry.

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