The Cuprizone mouse¹ is an established, repeatable model of demyelination that has been used to test many different MR methods that claim sensitivity to myelination state^2,3,4. Multi-component DESPOT (mcDESPOT⁵) is a fast method for imaging the Myelin Water Fraction (MWF), but to date has seen only a limited number of validation studies^6,7. It is hence a prime candidate for use with the Cuprizone mouse, and is interesting as most previous MR studies have been restricted to a limited number of slices and Regions-of-Interest for analysis^2,3,4, whereas mcDESPOT is a 3D method inherently capable of high-resolution full-brain imaging. Finally, a recent attempt to quantify the MWF in the Cuprizone mouse via Multi-Echo T2 failed even in healthy controls².

We imaged ex-vivo Cuprizone mice using mcDESPOT and looked for differences in T1, T2 and MWF across the whole brain using non-parametric statistics. We also acquired Diffusion Tensor Imaging (DTI) for comparison, particularly the Radial Diffusivity (RD) parameter which is often interpreted as a measure of myelin “integrity”, and then performed histology to confirm any findings.

8 control mice were fed powdered standard mouse diet while the food for another 8 mice was supplemented with 0.2% Cuprizone for 5 weeks. Mice were then sacrificed and transcardially perfused with 4% Paraformaldehyde (PFA). The heads were removed and immersed in PFA for 2 days before rehydration in Phosphate Buffered Saline for a minimum of 30 days. 2 controls and 2 Cuprizone heads at a time were then immersed in perfluorinated fluid (Galden, Solvay) to minimise susceptibility artefacts and scanned simultaneously. with a structural 3D Fast Spin-Echo, a mcDESPOT protocol with B1 correction consisting of SPGR, bSSFP & AFI⁷ scans, and a DTI scan. All scans had a 28.8mm FoV, other parameters were as follows. 3D Fast-Spin Echo: 256x256x256 matrix, TE/TR=40/3000 ms, scan-time 3 hours 25 minutes. SPGR: 192x192x192 matrix, TE/TR=5.1/20ms, flip-angles 4,5,6,8,10,12,16,20,24,26,28,30°, scan-time 2 hours 27 minutes. bSSFP: 192x192x192 matrix, TE/TR 3/6ms, flip-angles 8,9,10,12,15,20,30,40,50,55,60,65°, scan-time 2 hours 56 minutes. AFI: 96x96x96 matrix, TE/TR=4.3/20ms, flip-angle 55°, scan-time 40 minutes. Segmented EPI for DTI: 192x128 matrix, 40x0.5mm slices, TE/TR=43/4000ms, 4 segments, 30 diffusion directions with b=2000 & 4 b=0, scan-time 3 hours 7 minutes.

The T1, T2 & MWF maps were calculated using in-house open-source C++ software (github.com/spinicist/QUIT). DTI maps including RD were calculated using dtifit from FSL. A study template was built from the 3D FSE images using ANTs⁸, which was then registered to the Dorr mouse template⁹. All parameter maps were then transformed into the standard space. Non-parametric two-group T-tests were used to compare controls and Cuprizone mice using FSL randomise with the Threshold Free Cluster-Enhancement^10,11.

After scanning the brains were extracted and cryosectioned at 20µm and sections collected serially onto slides with 240µm between each section. Slides were then stained with Luxol Fast Blue for visualisation of Myelin content and with markers of microglia (Iba1) for neuroinflammation.

Figure 1 shows example slices through an MWF map produced by mcDESPOT in a control and Cuprizone mouse. Demyelination in the Corpus Callosum (CC) and in the Arbor Vitae of the Cerebellum is clearly visible and was confirmed in the histological sections. Figure 2 shows regions of increase in T1, T2, & RD and decrease in MWF between control and Cuprizone mouse thresholded at corrected $$$p < 0.05$$$. There is significant overlap but also some differences in these maps. In general, the T1 changes are the most focal and T2 most diffuse. RD shows increases in some different regions to the DESPOT measurements. Figure 3 is a rendering of the regions of decreased MWF, emphasising that Cuprizone causes widespread changes in the mouse brain that are not limited to the CC.mcDESPOT successfully measured the MWF in the Cuprizone mouse. Decreases are clearly visible in the CC but also in other regions including the Cerebellum. This demonstrates the power of quantitative, full-brain, high-resolution MRI and analysis methods compared to previous studies where only a limited number of ROIs have been used. Effects such as inflammation are often co-present with demyelination, and the slight differences in maps between T1, T2, MWF & RD suggest that although these are all sensitive to demyelination, they may not be specific.