Temporal Trajectories of Normal Myelination and Axonal Development Assessed by Quantitative Macromolecular and Diffusion MRI: Ultrastructural and Immunochemical Validation in a Rabbit Model

Introduction: Quantitative and non-invasive measures of brain myelination and maturation during development are of great importance to both clinical and translational research communities. While the metrics derived from diffusion tensor imaging, are sensitive to developmental changes and some pathologies, they remain difficult to relate to the actual microstructure of the brain tissue. The advent of advanced model-based microstructural metrics requires histological validation. The purpose of the study was to validate novel, model-based MRI techniques, such as macromolecular proton fraction mapping (MPF) and neurite orientation and dispersion indexing (NODDI), against histologically derived indexes of myelination and microstructural maturation at various stages of development.

Methods: New Zealand White rabbit kits underwent serial in-vivo MRI examination at postnatal days 1, 5, 11, 18, and 25, and as adults. Multi-shell, diffusion-weighted experiments were processed to fit NODDI model to obtain estimates, intracellular volume fraction (ICVF) and orientation dispersion index (ODI). Macromolecular proton fraction (MPF) maps were obtained from three source (MT-, PD-, and T1-weighted) images. After MRI sessions, a subset of animals was euthanized and regional samples of gray and white matter were taken for western blot analysis, to determine myelin basic protein (MBP), and electron microscopy, to estimate axonal, myelin fractions and g-ratio.

Results: MPF of white matter regions showed a period of fast growth between P5 and P11 in the internal capsule, with a later onset in the corpus callosum. This MPF trajectory was in agreement with levels of myelination in the corresponding brain region, as assessed by western blot and electron microscopy. In the cortex, the greatest increase of MPF occurred between P18 and P26. In contrast, myelin, according to MBP western blot, saw the largest hike between P5 and P11 in the sensorimotor cortex and between P11 and P18 in the frontal cortex, which then seemingly plateaued after P11 and P18 respectively. G-ratio by MRI markers decreased with age in the white matter. However, electron microscopy suggest a relatively stable g-ratio throughout development.

Conclusion: Developmental trajectories of MPF accurately reflected regional differences of myelination rate in different cortical regions and white matter tracts. MRI-derived estimation of g-ratio was inaccurate during early development, likely due to the overestimation of axonal volume fraction by NODDI due to the presence of a large proportion of unmyelinated axons.