Microstructural organization of axons in the human corpus callosum quantified by diffusion-weighted magnetic resonance spectroscopy of N-acetylaspartate and post-mortem histology

Diffusion-weighted magnetic resonance spectroscopy of brain metabolites offers unique access to compartment-specific microstructural information on neural tissue. Here, we investigated in detail the diffusion characteristics of the neuronal/axonal markers N-acetylaspartate + N-acetyl aspartyl glutamate (tNAA) in a small region of the human corpus callosum at 7 T. The diffusion-weighted spectroscopy data were analyzed by fitting to a model in which information about cross-callosal tract orientation within the spectroscopy volume, obtained from diffusion tensor imaging data, was incorporated. We estimated the microscopic misalignment of axons (σ _φ  = 18.6° ± 3.0°) in excellent agreement with independent histological results (σ _φ  = 18.1° ± 4.6°) obtained from microscopic analysis of axonal orientations in the body of the corpus callosum from post-mortem human brain slices. We also robustly quantified the diffusion coefficient of tNAA (0.51 ± 0.06 × 10⁻³ mm²/s) in axonal cytoplasm, unbiased by the tract curvature. This work supports the notion that microscopic axonal misalignment is a dominant microstructural property in white matter tracts and has a strong impact on the evaluation of tissue microstructure using diffusion information, and should therefore be taken into consideration in the evaluation of white matter microstructure. Additionally, this study enabled robust and unbiased assessment of the cytosolic diffusion coefficient of tNAA, a potential biomarker for axonopathy and neuronal degeneration.