Axon Diameters and Myelin Content Modulate Microscopic Fractional Anisotropy at Short Diffusion Times in Fixed Rat Spinal Cord

Mapping tissue microstructure accurately and noninvasively is one of the frontiers of biomedical imaging. Diffusion Magnetic Resonance Imaging (MRI) is at the forefront of such efforts, as it is capable of reporting on microscopic structures orders of magnitude smaller than the voxel size by probing restricted diffusion. Double Diffusion Encoding (DDE) and Double Oscillating Diffusion Encoding (DODE) in particular, are highly promising for their ability to report on microscopic fractional anisotropy (mu FA), a measure of the pore anisotropy in its own eigenframe, irrespective of orientation distribution. However, the underlying correlates of mu FA have insofar not been studied. Here, we extract mu FA from DDE and DODE measurements at ultrahigh magnetic field of 16.4T with the goal of probing fixed rat spinal cord microstructure. We further endeavor to correlate mu FA with Myelin Water Fraction (MWF) derived from multiexponential T-2 relaxometry, as well as with literature-based spatially varying axon diameter. In addition, a simple new method is presented for extracting unbiased mu FA from three measurements at different b-values. Our findings reveal strong anticorrelations between mu FA (derived from DODE) and axon diameter in the distinct spinal cord tracts; a moderate correlation was also observed between mu FA derived from DODE and MWF. These findings suggest that axonal membranes strongly modulate mu FA, which-owing to its robustness toward orientation dispersion effects-reflects axon diameter much better than its typical FA counterpart. mu FA varied when measured via oscillating or blocked gradients, suggesting selective probing of different parallel path lengths and providing insight into how those modulate mu FA metrics. Our findings thus shed light into the underlying microstructural correlates of mu FA and are promising for future interpretations of this metric in health and disease.