Assessing the Potential of Molecular Imaging for Myelin Quantification in Organotypic Cultures.

Pharmaceutics (2021-07-03)
Ander Egimendia, Susana Carregal-Romero, Iñaki Osorio-Querejeta, Daniel Padro, Jesús Ruiz-Cabello, David Otaegui, Pedro Ramos-Cabrer

Ex vivo models for the noninvasive study of myelin-related diseases represent an essential tool to understand the mechanisms of diseases and develop therapies against them. Herein, we assessed the potential of multimodal imaging traceable myelin-targeting liposomes to quantify myelin in organotypic cultures. Methods: MRI testing was used to image mouse cerebellar tissue sections and organotypic cultures. Demyelination was induced by lysolecithin treatment. Myelin-targeting liposomes were synthetized and characterized, and their capacity to quantify myelin was tested by fluorescence imaging. Results: Imaging of freshly excised tissue sections ranging from 300 µm to 1 mm in thickness was achieved with good contrast between white (WM) and gray matter (GM) using T2w MRI. The typical loss of stiffness, WM structures, and thickness of organotypic cultures required the use of diffusion-weighted methods. Designed myelin-targeting liposomes allowed for semiquantitative detection by fluorescence, but the specificity for myelin was not consistent between assays due to the unspecific binding of liposomes. Conclusions: With respect to the sensitivity, imaging of brain tissue sections and organotypic cultures by MRI is feasible, and myelin-targeting nanosystems are a promising solution to quantify myelin ex vivo. With respect to specificity, fine tuning of the probe is required. Lipid-based systems may not be suitable for this goal, due to unspecific binding to tissues.

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Triton X-100, for molecular biology
bisBenzimide H 33342 trihydrochloride, ≥98% (HPLC and TLC)
L-α-Lysophosphatidylcholine from egg yolk, ≥99%, Type I, powder
Goat serum
Bradford Reagent, for 0.1-1.4 mg/ml protein
Antibiotic Antimycotic Solution (100×), Stabilized, with 10,000 units penicillin, 10 mg streptomycin and 25 μg amphotericin B per mL, 0.1 μm filtered, BioReagent, suitable for cell culture
Agarose, BioReagent, for molecular biology, low EEO