Ischemic stroke is a serious public health problem associated with high mortality and morbidity rates [1]. It has been reported that approximately 15–20% of ischemic strokes are attributed to thromboembolism caused by carotid atherosclerosis [2], which is a progressive process. Lipids play a key role in the progression of atherosclerosis. They accumulate at sites of impaired endothelium in arterial walls, and subsequently activate inflammatory reactions and macrophages transformed from recruited monocytes in the intima and subintima. Foam cells are then formed through increased uptake of oxidized low-density lipoprotein and cause the initiation of atherosclerosis (fatty streaks) [3,4]. With continued accumulation of lipids and inflammatory cells, fatty streaks grow into atherosclerotic plaques characterized by lipid core surrounded by fibrous cap [5]. Atherosclerosis plaque continuously progresses into heterogeneous and complex structures (calcification, plaque fissures, hematoma and thrombus), and stroke events may eventually occur [6,7].

Macrophage phagocytize oxidized low-density lipoprotein (LDL), and transform into foam cells [8]. Uncontrolled uptake of LDL and impaired cholesterol efflux lead to the death of foam cells and accumulation of extracellular lipid droplets, causing the formation of lipid-rich regions (LRRs) [9]. Vascular smooth muscle cells (VSMCs) in the media layer can migrate to the sub-endothelial space and produce extracellular matrix proteins such as collagen and elastin forming collagen-rich regions (CRRs) [10]. Although previous studies showed that lipid profiles changed with the progression of atherosclerosis [11,12], the spatial distribution information was unavailable due to limited technology. Data on lipid profiles and spatial lipid patterns contextualized in terms of LRRs and CRRs, and their association with plaque progression remains unknown.

Spatial metabolomics is an emerging omics that can map the spatial distribution of small molecules, such as lipids, and correlate with pathological findings in situ without chemical labels or antibodies. Different spatial metabolomics technologies exist, such as time-of-flight secondary ion mass spectrometry (TOF-SIMS) and matrix-assisted laser desorption/ionization MSI (MALDI-MSI). In recent years, significant progress in this field has been achieved by desorption electrospray ionization MSI (DESI-MSI), which has the advantages of not destroying tissue sections, no matrix deposition, and to be performed under ambient conditions [13]. Previous studies have assessed atherosclerosis using TOF-SIMS [[14], [15], [16]], MALDI-MSI [[17], [18], [19], [20], [21]] and DESI-MSI [22] along various dimensions. However, only one study has reported on the spatial distribution of lipid in coronary artery atherosclerosis in a single patient by TOF-SIMS [16].

In this study, we employed DESI-MSI to investigate the spatial lipid profile distribution in human carotid plaques at different stages of atherosclerosis, and correlate MSI data with histological information, aiming to delineate metabolic profiles and provide deep insights into spatial metabolic mechanism of human carotid atherosclerosis.