Enhanced visualization of endocannabinoids spatial distribution in mouse brain via MALDI-2 mass spectrometry imaging

Endocannabinoids (eCBs) are endogenous lipid messengers that primarily bind cannabinoid receptors CB₁/CB₂ and together with the enzymes that regulate their biosynthesis and degradation define the endocannabinoid system. The eCB signaling system plays a key role in the central nervous system, and results often altered in neurological disorders. The analysis of eCBs is challenging due to their low concentration in biospecimens, and this is exacerbated in Mass Spectrometry Imaging (MSI) where low sensitivity and tissue dependent ion suppression obscure their spatial visualization. In this work we address this limitation by the application of laser-induced post-ionization (MALDI-2) approach. Herein we demonstrate that MALDI-2 boosts the detection of 2-arachidonylglycerol (2-AG) and N-acylethanolamines (AEA, PEA, OEA) with respect to MALDI, and that eCBs can be visualized in brain at physiological concentration only by MALDI-2-MSI. Root-mean-square (RMS), Total ion count (TIC) and internal standards (I.S.) normalization were evaluated, with I.S. normalization providing improved pixel to pixel variation and more uniform distribution for 2-AG and PEA in specific brain regions. Furthermore, high spatial resolution up to 5 μm pixel size was evaluated, resulting in the detection of all eCBs and confirming the MALDI-2 potential even reducing the ablated tissue amount. As proof of concept, the method was applied to map eCBs in a mouse model of mild traumatic brain injury, the APP-SWE mice, highlighting differences in the modulation of eCBs in Cortex, Hippocampus and Hypothalamus, suggesting the ability to reveal valuable biological insights for neuropharmacology.