MyDeepCARS

Current techniques of observation of myelin in vivo in the spinal cord axonal network, such as Magnetic resonance imaging and Positron emission tomography (PET), are limited in their specificity to myelin, and in their spatial scale resolution, which is not compatible with single cell scale observation. Understanding neurodegenerative diseases requires however a targeted in vivo observation of demyelination and neurodegeneration processes, at the molecular scale, and of their intricate relation over long time lapses. The goal of MyDeepCARS is to help clarifying the interaction between axons and their myelin sheaths in neurodegenerative diseases, using new optical methodologies. MyDeepCARS will develop in particular an optical microscope, based on Coherent Anti Stokes Raman Scattering (CARS) and two-photon fluorescence (2PF), to visualize myelin and axons in vivo, deep into spinal cord tissues, and to analyze the ultrastructure of myelin at the sub-micrometric scale. This tool will be capable of detecting the dynamics of early demyelination processes deep in the central nervous system of mice models. We will use four different models of demyelination; 1) neurodegeneration-induced-demyelination after rhizotomy 2) oligodendrocyte intoxication , 3) vascular endothelial growth factor (VEGF) triggered local immune attack, 4) MOG immunization induced multifocal experimental autoimmune encephalomyelitis (EAE), with the view not only to improve fundamental understanding of pathology progression but also to propose new tools for preclinical and clinical diagnosis. Based on the expertise present in the consortium on microscopy instrumentation, new microscopy modalities will be implemented, that do not exist yet in the literature of CARS imaging: 1) polarization resolved CARS microscopy (pCARS) to quantify the local lipids organization in myelin, in the spinal tracti of living mice; 2) wavefront shaping in-depth CARS imaging to correct for the sample optical distortions and scattering that prevent from reaching deeper regions below the superficial white matter tracts, and visualize also the spinal motor tracti that lay around 700 µm deep into the tissue. Taking advantage of transgenic mice with fluorescent axons implanted with a dorsal glass window developed in the consortium, we will evaluate by CARS/2PF microscopy the integrity of identified individual spinal cord axons submitted to local and multifocal demyelination processes over several days. The dynamics of axonal degeneration will be correlated to the number and extent of myelin damages and to the density of myelin weakness points identified as molecularly disorganized areas by pCARS, giving a unique way to evaluate the prevalence of demyelination over neurodegeneration and compare their relative progression at microscopic scale. Based on these observations, that should provide a new insight in the interaction that axons establish with their myelin sheath, we will evaluate the potential of the new pCARS technique as an early diagnostic tool for demyelinating pathologies, a field of high interest for the medical community. This project capitalizes on I. Fresnel’s developments on Coherent Anti Stokes Raman Scattering (CARS) for bio-imaging, and INT’s studies of the dynamics of cellular interactions in rodent models of Central Nervous System (CNS) pathologies. In this collaboration, both physicist and biologist skills will be hybridized into the development of new preclinical imaging modalities, designed to bring unprecedented level of information on the long-term development of neurodegenerative pathologies.