Proceedings of the National Academy of Sciences | 2021
Nile Red fluorescence spectroscopy reports early physicochemical changes in myelin with high sensitivity
Abstract
Significance Many diseases of the nervous system affect white matter and myelin, the critical lipid-rich insulating material enveloping axons. We report a method based on solvatochromic fluorescent probes and spectral microscopy that detects very early pathological changes in myelin in both a rodent model and in postmortem human multiple sclerosis brain at a stage that precedes overt histological myelin loss. Our method reveals that multiple sclerosis white matter exhibits widespread changes in myelin dielectric constant in areas distant from typical inflammatory demyelinating lesions. Fluorescence spectroscopy using solvatochromic lipid probes such as Nile Red has the ability to report, with high sensitivity and in a quantitative manner, early physicochemical changes in cells and tissues that precede pathological alterations detectable by conventional histological methods. 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.