The mechanical properties of living tissues have a significant impact on cell differentiation, but remain unexplored in the context of myelin formation and repair. In the PNS, the extracellular matrix (ECM) incorporates a basal lamina significantly denser than the loosely organized CNS matrix. Inhibition of non-muscle myosin II (NMII) enhances central but impairs peripheral myelination and NMII has been implicated in cellular responses to changes in the elasticity of the ECM. To directly evaluate whether mechanotransduction plays a role in glial cell differentiation, we cultured Schwann cells (SC) and oligodendrocytes (OL) on matrices of variable elastic modulus, mimicking either their native environment or conditions found in injured tissue. We found that a rigid, lesion-like matrix inhibited branching and differentiation of OL in NMII-dependent manner. By contrast, SC developed normally in both soft and stiffer matrices. Although SC differentiation was not significantly affected by changes in matrix stiffness alone, we found that expression of Krox-20 was potentiated on rigid matrices at high laminin concentration. These findings are relevant to the design of biomaterials to promote healing and regeneration in both CNS and PNS, via transplantation of glial progenitors or the implantation of tissue scaffolds.

Understanding the dynamic axon-glial cell interaction underlying myelination is hampered by the lack of suitable imaging techniques. Here we demonstrate third harmonic generation microscopy (THGM) for label-free imaging of myelinating Schwann cells in live culture and ex vivo and in vivo tissue. A 3D structure was acquired for a variety of compact and noncompact myelin domains, including juxtaparanodes, Schmidt-Lanterman incisures, and Cajal bands. Other subcellular features of Schwann cells that escape traditional optical microscopies were also visualized. We tested THGM for morphometry of compact myelin. Unlike current methods based on electron microscopy, g-ratio could be determined along an extended length of myelinated fiber in the physiological condition. The precision of THGMbased g-ratio estimation was corroborated in mouse models of hypomyelination. Finally, we demonstrated the feasibility of THGM to monitor morphological changes of myelin during postnatal development and degeneration. The outstanding capabilities of THGM may be useful for elucidation of the mechanism of myelin formation and pathogenesis.myelin | Schwann cell | multiphoton microscopy | label-free imaging | morphometry M yelin is a multiple-layered membrane sheath surrounding the axon. In myelinated nerves, the conduction of action potentials is much faster and the speed depends strongly on the structure of myelin. The structural integrity must be therefore tightly regulated for proper conduction of neuronal impulses, but the underlying axon-glial cell interaction is not well understood. Since the days of Ramón y Cajal, light microscopy has been widely used to visualize myelin morphology (1). A variety of fluorescent probes specifically binding to myelin components have allowed studies of the interaction between molecules (2-4). However, most such labeling methods are not suitable for unraveling in vivo dynamics of myelination because cell membranes are compromised during staining (especially immunohistochemistry) and/or the procedures are prohibitively timeconsuming and invasive. It is thus of great interest to develop label-free methods. Recently, spectral confocal reflectance microscopy has been demonstrated for high-resolution in vivo imaging of myelin (5). There are also techniques of nonlinear optical microscopy, which are generally known to be more advantageous for imaging deep live tissue. Coherence anti-Stokes Raman scattering (CARS) microscopy, which requires two synchronized short pulse lasers for excitation, has been used for imaging in vivo myelin and detecting pathology (6, 7). Third harmonic generation (THG) microscopy is based on another nonlinear optical process of light emission, yielding distinguishable images from CARS. Though it has been demonstrated for imaging the white matter in the brain (8), so far few studies have applied THG microscopy (THGM) for elucidating the mechanism of myelin formation. Moreover, the omission of the peripheral nervous systems (PNS) in the previous studies is not trivial considering the s...