Conventional MRI techniques have a very low sensitivity to gray matter demyelination. However, novel MRI techniques, such as magnetization transfer imaging, show potential to detect myelin content in gray matter¹. In this work we investigate whether diffusion-weighted MRI that is based on biophysical models of tissue microstructure^2,3 can detect demyelination in deep gray matter and cortex in the curpizone mouse model of MS.

Animals: A total of 20 female c57Bl/6 mice were divided randomly into 4 groups of 5 mice each. Three of the groups were exposed to cuprizone rich diet by adding 0.2% cuprizone to milled mouse chow and then sacrificed after 3 and 5 weeks of cuprizone exposure, and 4 weeks after ending the 5-week cuprizone exposure. The control mice were sacrificed at the end of the experimental period. Mice were euthanized by CO₂ asphyxiation, followed by intracardial perfusion with 4% formalin in PBS (phosphate-buffered saline). Brains were extracted, stored at 4^oC in 4% formalin, and then transferred to PBS 48 hours before scanning to remove formalin which degrades T₂ relaxation time. Finally, brains were placed into a custom-made acrylic holder and transferred to a 15 ml plastic tube that was filled with Fomblin oil (perfluoropolyether Y04 grade fluid).

MRI: Scanning was performed on a 7T horizontal-bore magnet using a head (23 mm ID) quadrature volume resonator (both from Bruker Corporation, Germany). Diffusion-weighted EPIs were acquired using a Stejskal-Tanner spin-echo diffusion preparation. Imaging parameters were: 128x128 data matrix, resolution 98μmx98μm, slice thickness 0.75 mm, TR/TE=3s/33.49 ms, N_avg=6. Total scan time was 3 hours 18 min. Diffusion parameters were: δ/Δ = 6 ms/20 ms. Sixteen b-factors ranging linearly from 880 to 14080 s/mm², 10 diffusion directions, and five A₀ images were acquired.

Analysis: Images were analysed in MATLAB (R_2013a, MathWorks) using Jespersen’s et al.^2,3 dendrite density model of gray matter. Their biophysical model of tissue microstructure assumes that the MR diffusion signal originates from two components: (i) the dendrites and axons (i.e. neurites), modeled as long cylinders with one diffusion coefficient parallel (D_L) to the cylindrical axis (D_T=0 due to the diffusion resolution limit), and (ii) an isotropic mono-exponential diffusion component (D_ext) describing water diffusion within extracellular space. The results of the fit were parametric maps of neurite density, D_L and D_ext. Region-of-interest (ROI) analysis was performed by selecting ROIs in the right CX and in DGM within a slice which was the same for all animals. Mean values within the ROIs were recorded and used to compute the population mean and standard error. Wilcoxon rank sum test was used to compute significant differences between groups.

Figure 1 shows parameteric maps of relative neurite density for one representative animal within each group. There is a visible reduction in neurite density in DGM and CX after 5 weeks of exposure, and remyelination 4 weeks after terminating the exposure. Figure 2 shows mean values of neurite density, D_ext and D_L from ROI analysis of CX and DGM for 5 animals in each of the groups and 95% confidence intervals. We detected significant differences (p<0.05) in the values of neurite density and D_L between baseline and at 3 and 5 weeks of cuprizone exposure in both DGM and CX. After 3 weeks of exposure, neurite density decreased by 16% and D_L increased by app 20%. There were no significant differences in values between 3 and 5 weeks of exposure. Both, the neurite density and D_L returned to the baseline values (0.5 and 0.8-1.0 μm²/ms, respectively) after remyelination. Extracellular diffusion in DGM and CX did not change significantly as a result of cuprizone exposure and had mean values between 0.27 and 0.35 μm²/ms for all experimental groups. Our measurements showed that the neurite volume fraction decreased while the longitudinal diffusion coefficient increased with cuprizone exposure. Myelin presents a barrier to spin motion so when myelin is lost, spins can move more freely in all directions, decreasing diffusion anisotropy and increasing diffusion coefficient. On the contrary, the extracellular diffusivity did not change significantly with cuprizone exposure. This could be understood in light of the fact that the extracellular space and cell bodies occupy only approximately 18% of mouse cortex, so any changes in extracellular diffusion coefficient, which is in addition small due to highly restricted extracellular space, will not be easily detectable.

In this work we have demonstrated that the degree of demyelination in the cuprizone mouse model of MS correlates well with the neurite density and intra-axonal diffusion parameter of the Jespersen's dendrite density model and therefore could serve as markers of demyelination.