Nile Red fluorescence spectroscopy reports early physicochemical changes in myelin with high sensitivity

The molecular composition of myelin membranes determines their structure and function. Even minute changes to the biochemical balance can have profound consequences for axonal conduction and the synchronicity of neural networks. Hypothesizing that the earliest indication of myelin injury involves changes in the composition and/or polarity of its constituent lipids, we developed a sensitive spectroscopic technique for defining the chemical polarity of myelin lipids in fixed frozen tissue sections from rodent and human. The method uses a simple staining procedure involving the lipophilic dye Nile Red, whose fluorescence spectrum varies according to the chemical polarity of the microenvironment into which the dye embeds. Nile Red spectroscopy identified histologically intact yet biochemically altered myelin in prelesioned tissues, including mouse white matter following subdemyelinating cuprizone intoxication, as well as normal-appearing white matter in multiple sclerosis brain. Nile Red spectroscopy offers a relatively simple yet highly sensitive technique for detecting subtle myelin changes.

Myelin is a highly ordered, lipid-rich extension of glial cell membrane that facilitates rapid and efficient saltatory conduction of action potentials along axons in the central and peripheral nervous systems. The stability of myelin membranes critically depends on its molecular composition (1–3). Although myelin is maintained roughly at a ratio of 70:30% lipid to protein (4), lipid membranes are highly fluid; changes in lipid composition are defining characteristics of myelin development (5), homeostasis in the adult, and aging in rodents (6, 7), as well as primates (8). Shifts in lipid composition also occur in inflammatory demyelinating disorders like multiple sclerosis (MS) (9, 10). Lipids are even theorized to be targets of immune attacks in autoimmune disorders, a role previously ascribed to proteins (11). Key roles for lipids notwithstanding, tools to interrogate biochemical changes to myelin lipids have largely been restricted to in vitro systems.Once thought to be inert, myelin is now known to be a chemically and structurally dynamic element (12). Specific combinations of proteins and lipids induce formation and compaction of multilamellar vesicles that resemble myelin (13), underscoring the importance of correct chemical composition for assembly. Conversely, alterations in these molecular proportions promote decompaction and myelin vesiculation (3, 14). The polarity of lipid species in cell membranes influences their packing properties and therefore stability (15). Governed by competing thermodynamic forces of lipid curling and hydrocarbon packing (16), myelin sheaths lie at the critical edge of bilayer stability and thus are susceptible to factors in the environment. Indeed, the myelin integrity theory of MS rests on the outsized influence of environmental forces on myelin stability and function (17). Therefore, methods for detecting physicochemical changes in myelin lipid composition in situ would greatly enhance our understanding of early events in myelin development, as well as myelin damage in disease states, with important implications for therapies designed to prevent myelin loss in MS and other demyelinating disorders.The study of myelin lipid biochemistry poses unique challenges (18). Traditional analytical methods, such as thin-layer chromatography and high-performance liquid chromatography (19), depend on tissue homogenization that eliminates informative spatial relationships. Imaging lipid mass spectrometry (20) preserves spatial relationships, but submicron resolution has yet to be realized, and reproducibility at the level of sample preparation remains problematic (21). Coherent anti–Stokes Raman scattering microscopy provides high-resolution, label-free imaging of lipids in histological samples (22), but this method lacks sensitivity and requires expertise in nonlinear optics as well as highly specialized hardware. Finally, fluorescent lipophilic dyes, though widely available and easy to use, have traditionally been employed to detect lipid-rich structures in only a qualitative manner. Conventional fluorescence microscopy is therefore unable to detect subtle shifts in lipid biochemistry. By contrast, Nile Red (NR) is a fluorescent dye that is well situated to report changes in the chemical polarity of cell membranes and myelin, being both lipophilic (23, 24) and differentially fluorescent depending on solvent environment (i.e., exhibits solvatochromism) (25). The current study uses NR fluorescence spectroscopy to identify polarity shifts as an early manifestation of myelin disease prior to overt demyelination. We show that this technique reports subtle biochemical changes in myelin, resulting in a method that is a very sensitive marker of incipient myelin injury.