Abstract
Analyzing lipid composition and distribution within the brain is important to study white matter pathologies that present focal demyelination lesions, such as multiple sclerosis. Some lesions can endogenously re-form myelin sheaths. Therapies aim to enhance this repair process in order to reduce neurodegeneration and disability progression in patients. In this context, a lipidomic analysis providing both precise molecular classification and well-defined localization is crucial to detect changes in myelin lipid content. Here we develop a correlated heterospectral lipidomic (HSL) approach based on coregistered Raman spectroscopy, desorption electrospray ionization mass spectrometry (DESI-MS), and immunofluorescence imaging. We employ HSL to study the structural and compositional lipid profile of demyelination and remyelination in an induced focal demyelination mouse model and in multiple sclerosis lesions from patients ex vivo. Pixelwise coregistration of Raman spectroscopy and DESI-MS imaging generated a heterospectral map used to interrelate biomolecular structure and composition of myelin. Multivariate regression analysis enabled Raman-based assessment of highly specific lipid subtypes in complex tissue for the first time. This method revealed the temporal dynamics of remyelination and provided the first indication that newly formed myelin has a different lipid composition compared to normal myelin. HSL enables detailed molecular myelin characterization that can substantially improve upon the current understanding of remyelination in multiple sclerosis and provides a strategy to assess remyelination treatments in animal models.
Highlights
Multiple sclerosis is a degenerative condition of the central nervous system, characterized by multifocal inflammatory demyelinating lesions
We developed a new correlated heterospectral lipidomics (HSL) imaging strategy for molecular characterization and quantification of remyelination based on correlative Raman spectroscopy and desorption electrospray ionization mass spectrometry (DESI-mass spectrometric (MS)) analysis, validated with immunolabeling for specific myelin proteins
The significant increase in DAPI+ cells observed at day 14 after LPC injection (1.4 fold increase ± 0.13, p < 0.05) compared to control corresponds to an accumulation of microglia/macrophages in the lesion, which we identified by correlative immunofluorescence for the microglial marker IBA1 in a subset of sections already analyzed by Raman spectroscopy (Supplementary Figure S4)
Summary
Multiple sclerosis is a degenerative condition of the central nervous system, characterized by multifocal inflammatory demyelinating lesions. To find an effective pro-remyelinating therapy, it will be crucial to reliably and quickly analyze the amount of myelination in different lesions at the molecular level in animal models of demyelination and remyelination and, ideally, in living multiple sclerosis patients. To detect and quantify remyelination in tissue samples, whether from animal models or multiple sclerosis patient brain tissue samples, researchers generally use histochemical staining (e.g., Luxol fast blue staining), or immunohistochemistry using antibodies targeted against myelin proteins such as myelin basic protein (MBP), or proteolipid protein,[10] but do not assess lipids which are the main component of myelin. The current gold standard for more comprehensive quantification of remyelination in a lesion, especially in animal models of demyelination, is to use electron microscopy to directly observe the number of myelinated axons and myelin sheath thickness, as remyelinated myelin is usually thinner than normal myelin.[11,12] This method, does not examine the molecular content.
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