Lipid order degradation in autoimmune demyelination probed by polarization resolved coherent Raman microscopy

Myelin around axons is currently widely studied by structural analyses and large scale imaging techniques, with the goal to decipher its critical role in neuronal protection. While there is strong evidence that in myelin, lipid composition and lipid membrane morphology are affected during the progression of neurodegenerative diseases, there is no quantitative method yet to report its ultrastructure in tissues at both molecular and macroscopic levels, in conditions potentially compatible with in vivo observations. In this work, we study and quantify molecular order of lipids in myelin at sub-diffraction scales, using label-free polarization resolved Coherent Anti Stokes Raman (PR-CARS), which exploits CARS sensitivity to coupling between light polarization and oriented molecular vibrational bonds. Importantly, the method does not use any a priori parameters in the sample such as lipid type, orientational organization and composition. We show that lipid molecular order of myelin in the mouse spinal cord is significantly reduced throughout the progression of experimental autoimmune encephalomyelitis (EAE), a model for multiple sclerosis, even in myelin regions that appear morphologically unaffected. This technique permits to unravel molecular-scale perturbations of lipid layers at early stage of the demyelination progression, while the membrane architecture at the mesoscopic scale (here about 100 nm) seems much less affected. Such information cannot be brought by pure morphological observation and opens new prospectives towards molecular-scale understanding of neurodegenerative diseases.