Spatial segmentation and metabolite annotation involved in sperm maturation in the rat epididymis by MALDI imaging mass spectrometry.

Spermatozoa acquire their fertilizing capacity during a complex maturation process that occurs in the epididymis. This process involves a substantial molecular remodeling at the surface of the gamete. Epididymis is divided into three regions (the caput, corpus, and cauda) or into 19 intraregional segments based on histology. Most studies carried out on epididymal maturation have been performed on sperm samples or tissue extracts. Here, we used MALDI imaging mass spectrometry in the positive and negative ion modes combined with spatial segmentation and automated metabolite annotation to study the precise localization of metabolites directly in the rat epididymis. The spatial segmentation revealed that the rat epididymis could be divided into several molecular clusters different from the 19 intraregional segments. The discriminative m/z values that contributed the most to each molecular cluster were then annotated and corresponded mainly to phosphatidylcholines, sphingolipids, glycerophosphates, triacylglycerols, plasmalogens, phosphatidylethanolamines, and lysophosphatidylcholines. A substantial remodeling of lipid composition during epididymal maturation was observed. It was characterized in particular by an increase in the number of sphingolipids and plasmalogens and a decrease in the proportion of triacylglycerols annotated from caput to cauda. Ion images reveal that molecules belonging to the same family can have very different localizations along the epididymis. For some of them, annotation was confirmed by on-tissue MS/MS experiments. A 3D model of the epididymis head was reconstructed from 61 sections analyzed with a lateral resolution of 50 µm and can be used to obtain information on the localization of a given analyte in the whole volume of the tissue.

Three-Dimensional Mass Spectrometry Imaging Identifies Lipid Markers of Medulloblastoma Metastasis.

Treatment for medulloblastoma (MB) - the most common malignant pediatric brain tumor - includes prophylactic radiation administered to the entire brain and spine due to the high incidence of metastasis to the central nervous system. However, the majority of long-term survivors are left with permanent and debilitating neurocognitive impairments as a result of this therapy, while the remaining 30-40% of patients relapse with terminal metastatic disease. Development of more effective targeted therapies has been hindered by our lack of understanding of the underlying mechanisms regulating the metastatic process in this disease. To understand the mechanism by which MB metastasis occurs, three-dimensional matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) experiments were performed on whole brains from a mouse model of human medulloblastoma. Analyzing the tumor and surrounding normal brain in its entirety enabled the detection of low abundance, spatially-heterogeneous lipids associated with tumor development. Boundaries of metastasizing and non-metastasizing primary tumors were readily defined, leading to the identification of lipids associated with medulloblastoma metastasis, including phosphatidic acids, phosphatidylethanolamines, phosphatidylserines, and phosphoinositides. These lipids provide a greater insight into the metastatic process and may ultimately lead to the discovery of biomarkers and novel targets for the diagnosis and treatment of metastasizing MB in humans.

Proteomic Analysis of the Spatio-temporal Based Molecular Kinetics of Acute Spinal Cord Injury Identifies a Time- and Segment-specific Window for Effective Tissue Repair.

Spinal cord injury (SCI) represents a major debilitating health issue with a direct socioeconomic burden on the public and private sectors worldwide. Although several studies have been conducted to identify the molecular progression of injury sequel due from the lesion site, still the exact underlying mechanisms and pathways of injury development have not been fully elucidated. In this work, based on OMICs, 3D matrix-assisted laser desorption ionization (MALDI) imaging, cytokines arrays, confocal imaging we established for the first time that molecular and cellular processes occurring after SCI are altered between the lesion proximity, i.e. rostral and caudal segments nearby the lesion (R1-C1) whereas segments distant from R1-C1, i.e. R2-C2 and R3-C3 levels coexpressed factors implicated in neurogenesis. Delay in T regulators recruitment between R1 and C1 favor discrepancies between the two segments. This is also reinforced by presence of neurites outgrowth inhibitors in C1, absent in R1. Moreover, the presence of immunoglobulins (IgGs) in neurons at the lesion site at 3 days, validated by mass spectrometry, may present additional factor that contributes to limited regeneration. Treatment in vivo with anti-CD20 one hour after SCI did not improve locomotor function and decrease IgG expression. These results open the door of a novel view of the SCI treatment by considering the C1 as the therapeutic target.