Raman Probes for In Situ Molecular Analyses of Peripheral Nerve Myelination

The myelinating activity of living Schwann cells in coculture with neuronal cells was examined in situ in a Raman microprobe spectroscope. The Raman label-free approach revealed vibrational fingerprints directly related to the activity of Schwann cells' metabolites and identified molecular species peculiar to myelinating cells. The identified chemical species included antioxidants, such as hypotaurine and glutathione, and compartmentalized water, in addition to sphingolipids, phospholipids, and nucleoside triphosphates also present in neuronal and nonmyelinating Schwann cells. Raman maps at specific frequencies could be collected, which clearly visualized the myelinating action of Schwann cells and located the demyelinated ones. An important finding was the spectroscopic visualization of confined water in the myelin structure, which exhibited a quite pronounced Raman signal at ∼3470 cm^-1. This peculiar signal, whose spatial location precisely corresponded to a low-frequency fingerprint of hypotaurine, was absent in unmyelinating cells and in bulk water. Raman enhancement was attributed to frustration in the hydrogen-bond network as induced by interactions with lipids in the myelin sheaths. According to a generally accepted morphological model of myelin, an explanation was offered of the peculiar Raman scattering of water confined in intraperiod lines, according to an ordered hydrogen bonding structure. The possibility of concurrently mapping antioxidant molecules and compartmentalized water structure with high spectral accuracy and microscopic spatial resolution enables probing myelinating activity and might play a key-role in future studies of neuronal pathologies. Compatible with life, Raman microprobe spectroscopy with the newly discovered probes could be suitable for developing advanced strategies in the reconstruction of injured nerves and nerve terminals at neuromuscular junctions.