MALDI-2 Mass Spectrometry Imaging enables spatial visualization of endocannabinoids in brain

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, resulting altered in most neurological disorders. The analysis of eCBs is challenging for 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). 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 endogenous concentration only by MALDI-2-MSI. Both root-mean-square (RMS) and deuterated internal standards (I.S.) normalization were evaluated, with I.S. normalization providing improved pixel to pixel variation and more uniform distribution in specific brain regions, especially for 2-AG and PEA. Furthermore, high lateral 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. Lastly the method was applied as proof of concept in a mouse model of mild traumatic brain injury demonstrating the ability to reveal valuable biological insights for neuropharmacology.