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

Abstract

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.

Proteomics of Multiple Sclerosis: Inherent Issues in Defining the Pathoetiology and Identifying (Early) Biomarkers

“…This strongly supports the previous proposal that metabolic dysregulation leads to the degeneration of oligodendrocytes [ 10 , 104 ]. Importantly, these metabolic changes are detected in CPZ-fed mice prior to the detection of any overt demyelination by conventional tools, consistent with biochemical changes preceding oligodendrocyte degeneration and subsequent demyelination [ 13 ]. However, most proteins (~80%) do not exert their biological functions alone but rather work with other proteins [ 249 , 250 ].…”

“…However, the data summarised here make it clear that the advent and extensive adoption of shotgun analysis has not promoted a deeper understanding of underlying, initiating mechanisms, nor led to the identification of definitive (early) biomarkers. Nonetheless, overall, metabolically dysregulated proteins were found as a major cluster across animal models and MS, suggesting that some as yet undefined internal metabolic dysfunction in oligodendrocytes (or other local cells such as astrocytes) may initiate MS [ 12 , 13 ]. Overall, we conclude that: Differential changes occur in protein abundance irrespective of whether the samples analysed are from MS patients or animal models.…”

“…N-acetylaspartate, a metabolite of aspartic acid, is highly expressed in neuronal and glial cells and its reduction indicates axonal injury and is correlated with disease severity in RRMS patients [ 20 , 23 ]. Consistent with the early metabolic changes preceding clinical diagnosis of MS [ 21 ], a recent report using Nile Red fluorescence spectroscopy revealed subtle biochemical alterations in the lipid constituents (i.e., metabolic changes) of histologically intact (i.e., pre-lesion) medial corpus callosum after only two days of feeding cuprizone (CPZ) to mice [ 13 ]. A similar change was observed in normal-appearing white and grey matter from MS patients [ 13 ].…”

“…Consistent with the early metabolic changes preceding clinical diagnosis of MS [ 21 ], a recent report using Nile Red fluorescence spectroscopy revealed subtle biochemical alterations in the lipid constituents (i.e., metabolic changes) of histologically intact (i.e., pre-lesion) medial corpus callosum after only two days of feeding cuprizone (CPZ) to mice [ 13 ]. A similar change was observed in normal-appearing white and grey matter from MS patients [ 13 ]. Notably, using traditional histological staining methods (e.g., Luxol-fast blue), it takes 2–5 weeks of CPZ-feeding to detect measurable changes [ 24 ].…”

“…In the CNS, these immune cells contribute to a complex chronic neuro-inflammatory state, oligodendrocyte degeneration or death (i.e., oligodendrocytopathy or oligodendrocytosis), demyelination and neuronal loss [ 6 , 7 , 8 ]. However, other biochemical changes in the CNS may well precede the involvement of the peripheral immune system [ 9 , 10 , 11 , 12 , 13 ]. Based on the clinical diagnostic criteria, MS is categorized into four broad phenotypes: primary progressive MS (PPMS), secondary progressive MS (SPMS), relapsing-remitting MS (RRMS), and progressive relapsing MS (PRMS; [ 7 , 14 , 15 ]).…”