CARS Imaging to Evaluate Neurodegenerative and Demyelinating Activity

Non-linear optical microscopy offers the potential for label free methods to image and evaluate tissue function and subcellular responses. Coherent Anti-Stokes Raman Scattering (CARS) permits label-free imaging of structures using vibrational contrast inherent in the sample. Here we present the application of CARS imaging to evaluate (sub)cellular changes in neurodegenerative and demyelinating activity in animal models and in vitro. Towards these ends we evaluate cellular changes in the experimental autoimmune encephalomyelitis (EAE) and cuprizone models of demyelination as well as in a 3D printed synthetic myelination assay platform. Multiplexed CARS signals within individual cellular subtypes, myelin imaging and second harmonic generation (SHG) were used to identify degenerative pathology, treatment response and evaluated using novel computational techniques. Myelination is characterized by acquiring the highly specialized myelin membrane around axons. The process is particularly active during early years continuing into adolescence, and it plays a crucial role in the maturation and proper functioning of the nervous system. During disease states such as MS, Parkinson's [1], Schizophrenia [2], depression [3], and Alzheimer's [4], demyelination is usually observed through damage caused to the protective cover (myelin sheath) that surrounds axons in the spinal cord, fibers in the brain and the optic nerves. We evaluate dynamic changes in astrocytes and oligodendrocytes in two mouse demyelination models, EAE and cuprizone [5] mediated toxicity, as assessed by CARS microscopy. CARS myelin and SHG imaging clearly identifies structural changes in tissue, treatment effects and spectral imaging coupled with computational approaches indicates potential signatures of cellular subtypes associated with disease. Further, to evaluate the use of specific treatments to stimulate myelination, and the utility of CARS imaging in vitro, we show proof-of-concept data validating our novel printed liquid crystal elastomer axonal cell culture myelination assay. CARS microscopy offers a relatively simple non-invasive method to probe neurodegenerative and demyelinating activity, and coupled new computational algorithms offers promise to decode subcellular activities. CARS microscopy is a unique, expandable tool to evaluate cellular function without the limitations of fluorophores and staining protocols compatible with live cultures.