Characterization of Humanized Mouse Model of Organophosphate Poisoning and Detection of Countermeasures via MALDI-MSI.

Organophosphoate (OP) chemicals are known to inhibit the enzyme acetylcholinesterase (AChE). Studying OP poisoning is difficult because common small animal research models have serum carboxylesterase, which contributes to animals' resistance to OP poisoning. Historically, guinea pigs have been used for this research; however, a novel genetically modified mouse strain (KIKO) was developed with nonfunctional serum carboxylase (Es1 KO) and an altered acetylcholinesterase (AChE) gene, which expresses the amino acid sequence of the human form of the same protein (AChE KI). KIKO mice were injected with 1xLD50 of an OP nerve agent or vehicle control with or without atropine. After one to three minutes, animals were injected with 35 mg/kg of the currently fielded Reactivator countermeasure for OP poisoning. Postmortem brains were imaged on a Bruker RapifleX ToF/ToF instrument. Data confirmed the presence of increased acetylcholine in OP-exposed animals, regardless of treatment or atropine status. More interestingly, we detected a small amount of Reactivator within the brain of both exposed and unexposed animals; it is currently debated if reactivators can cross the blood-brain barrier. Further, we were able to simultaneously image acetylcholine, the primary affected neurotransmitter, as well as determine the location of both Reactivator and acetylcholine in the brain. This study, which utilized sensitive MALDI-MSI methods, characterized KIKO mice as a functional model for OP countermeasure development.

Mass Spectrometry Imaging of Organoids to Improve Preclinical Research.

Preclinical models are essential research tools before novel therapeutic or diagnostic methods can be applied to humans. These range from in vitro cell monocultures to vastly more complex animal models, but clinical translation to humans often fails to deliver significant results. Three-dimensional (3D) organoid systems are being increasingly studied to establish physiologically relevant in vitro platforms in a trade-off between the complexity of the research question and the complexity of practical experimental setups. The sensitivity and precision of analytical tools are yet another limiting factors in what can be investigated, and mass spectrometry (MS) is one of the most powerful analytical techniques available to the scientific community. Its innovative use to spatially resolve biological samples has opened many research avenues in the field of MS imaging (MSI). Here, this work aims to explore the current scientific landscape in the application of MSI on organoids, with an emphasis on their combined potential to facilitate and improve preclinical studies.

FluoMALDI Microscopy: Matrix Co-Crystallization Simultaneously Enhances Fluorescence and MALDI Imaging.

Here, the authors report that co-crystallization of fluorophores with matrix-assisted laser desorption/ionization (MALDI) imaging matrices significantly enhances fluorophore brightness up to 79-fold, enabling the amplification of innate tissue autofluorescence. This discovery facilitates FluoMALDI, the imaging of the same biological sample by both fluorescence microscopy and MALDI imaging. The approach combines the high spatial resolution and specific labeling capabilities of fluorescence microscopy with the inherently multiplexed, versatile imaging capabilities of MALDI imaging. This new paradigm simplifies registration by avoiding physical changes between fluorescence and MALDI imaging, allowing to image the exact same cells in tissues with both modalities. Matrix-fluorophore co-crystallization also facilitates applications with insufficient fluorescence brightness. The authors demonstrate  feasibility of FluoMALDI imaging with endogenous and exogenous fluorophores and autofluorescence-based FluoMALDI of brain and kidney tissue sections. FluoMALDI will advance structural-functional microscopic imaging in cell biology, biomedicine, and pathology.

RaMALDI: Enabling simultaneous Raman and MALDI imaging of the same tissue section.

Multimodal tissue imaging techniques that integrate two complementary modalities are powerful discovery tools for unraveling biological processes and identifying biomarkers of disease. Combining Raman spectroscopic imaging (RSI) and matrix-assisted laser-desorption/ionization (MALDI) mass spectrometry imaging (MSI) to obtain fused images with the advantages of both modalities has the potential of providing spatially resolved, sensitive, specific biomolecular information, but has so far involved two separate sample preparations, or even consecutive tissue sections for RSI and MALDI MSI, resulting in images with inherent disparities. We have developed RaMALDI, a streamlined, integrated, multimodal imaging workflow of RSI and MALDI MSI, performed on a single tissue section with one sample preparation protocol. We show that RaMALDI imaging of various tissues effectively integrates molecular information acquired from both RSI and MALDI MSI of the same sample, which will drive discoveries in cell biology, biomedicine, and pathology, and advance tissue diagnostics.

Current Knowledge on Intestinal Absorption of Anthocyanins.

Anthocyanins are flavonoid compounds that are natural color pigments occurring in various colored plants, such as berry fruits, vegetables, and grapes. With the elucidation of their various physiological effects, anthocyanins have been identified as promising functional food ingredients. However, findings on the bioavailability of anthocyanins, which are present in various chemical structures in foods, are limited; their intestinal absorption behaviors, including their transport route(s), have not been fully explained. This perspective overviews the current knowledge and issues and discusses advanced techniques, such as in situ matrix-assisted laser desorption/ionization mass spectrometry imaging, and future perspectives on the study of the bioavailability of anthocyanins.

Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Imaging of Tissues via the Formation of Reproducible Matrix Crystals by the Fluorescence-Assisted Spraying Method: A Quantification Approach.

The application of matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) imaging to quantitative analyses is restricted by the variability of MS intensity of the analytes in nonreproducible matrix crystals of tissues. To overcome this challenge, a fluorescence-assisted spraying method was developed for a constant matrix amount employing an MS-detectable fluorescent reagent, rhodamine 6G (R6G), which was sprayed with the matrix. To form a homogeneous matrix crystal on the tissue section, a matrix solution, 1,5-diaminonaphthalene (10 mg/mL), containing R6G (40 µg/mL) and O-dinitrobenzene (O-DNB, 10 mg/mL) was sprayed until the desired constant fluorescence intensity was achieved. Compared with that obtained via conventional cycle-number-fixed spraying [relative standard deviation (RSD) = 31.1%], the reproducibility of the relative MS intensity of the analyte [ferulic acid (FA), RSD = 3.1%] to R6G was significantly improved by the fluorescence-assisted matrix spraying. This result indicated that R6G could be employed as an index of the matrix amount and an MS normalizing standard. The proposed matrix spraying successfully quantified nifedipine (0.5-40 pmol/mm2 in the positive mode, R2 = 0.965) and FA (0.5-75 pmol/mm2 in the negative mode, R2 = 0.9972) in the kidney section of a rat. Employing the quantitative MALDI-MS imaging assay, FA, which accumulated in the kidney of the rat after 50 mg/kg was orally administered, was visually determined to be 3.5, 3.0, and 0.2 µmol/g tissue at 15, 30, and 60 min, respectively.

Accumulation of Plasma-Derived Lipids in the Lipid Core and Necrotic Core of Human Atheroma: Imaging Mass Spectrometry and Histopathological Analyses.

Objective: To clarify the pathogenesis of human atheroma, the origin of deposited lipids, the developmental mechanism of liponecrotic tissue, and the significance of the oxidation of phospholipids were investigated using mass spectrometry-aided imaging and immunohistochemistry.Atherosclerotic lesions in human coronary arteries were divided into 3 groups: pathologic intimal thickening with lipid pool, atheroma with lipid core, and atheroma with necrotic core. The lipid pool and lipid core were characterized by the deposition of extracellular lipids. The necrotic core comprised extracellular lipids and liponecrotic tissue. The proportion of cholesteryl linoleate in cholesteryl linoleate+cholesteryl oleate fraction in the extracellular lipid and liponecrotic regions differed significantly from that of the macrophage foam cell-dominant region, and the plasma-derived components (apolipoprotein B and fibrinogen) were localized in the regions. The liponecrotic region was devoid of elastic and collagen fibers and accompanied by macrophage infiltration in the surrounding tissue. Non-oxidized phospholipid (Non-OxPL), OxPL, and Mox macrophages were detected in the three lesions. In the atheroma with lipid core and atheroma with necrotic core, non-OxPL tended to localize in the superficial layer, whereas OxPL was distributed evenly. Mox macrophages were colocalized with OxPL epitopes.In human atherosclerosis, plasma-derived lipids accumulate to form the lipid pool of pathologic intimal thickening, lipid core of atheroma with lipid core, and necrotic core of atheroma with necrotic core. The liponecrotic tissue in the necrotic core appears to be developed by the loss of elastic and collagen fibers. Non-OxPL in the accumulated lipids is oxidized to form OxPL, which may contribute to the lesion development through Mox macrophages.

Matrix-assisted laser desorption/ionization mass spectrometry-guided visualization analysis of intestinal absorption of acylated anthocyanins in Sprague-Dawley rats.

It is unknown whether intestinal absorption of acylated anthocyanins occurs in their intact or metabolized form. In this study, with the aid of matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) imaging, intestinal absorption of acylated anthocyanins was visually investigated. Anthocyanin extracts from purple carrots were orally administered to Sprague-Dawley rats. Acylated cyanidins were absorbed into portal and circulating blood systems in their intact form, and aglycon; cyanidin 3-O-(6-O-feruloyl-ß-d-glucopyranosyl)-(1 â†’ 6)-[ß-d-xylopyranosyl-(1 â†’ 2)]-ß-d-galactopyranoside (Cy3XFGG), and showed a high absorption of 39.3 ± 0.1 pmol/mL-plasma at 60 min after administration. MALDI-MS imaging analysis of the rat jejunum membranes showed that an organic anion transporting polypeptide (OATP) transporter was involved in Cy3XFGG transport, while deacylated anthocyanins were incorporated through both the glucose transporter 2 and OATP routes. In conclusion, acylated anthocyanin, Cy3XFGG, can be absorbed in its intact form through intestinal OATP.