Monitoring Demyelination in a Cuprizone Mouse Model with Longitudinal and Quantitative MRI Measurements

Introduction Magnetic resonance imaging (MRI) methods capable of quantifying changes due to demyelination can improve both the diagnosis and understanding of white matter diseases such as multiple sclerosis. T2-weighted (T2w) and magnetization transfer images (MTI) were acquired weekly in control (n=4) and cuprizone-fed mice (n=4). Diffusion tensor imaging (DTI), quantitative MTI, high-resolution T2w imaging, and histopathology were used to analyze ex vivo tissue. All in vivo methods showed significant differences longitudinally in the corpus callosum (CC) of the cuprizone-fed mouse. All in vivo and ex vivo methods showed significant differences in the corpus callosum between groups. Methods Mouse Model: Cuprizone is a toxic model of demyelination, causing cell death of oligodendrocytes followed by extensive demyelination. C57BL/6 mice were fed 0.4% cuprizone (w/w) starting at 8 weeks of age. After 6 weeks of feeding, mice were perfused with PBS/saline solution followed by 4% PFA. Heads were fixed for 24 hours in 4% PFA, after which all tissue was removed external to the skull. 48 hours prior to ex vivo imaging, brains were transferred to a PBS solution to leach out the remaining PFA. All experiments were approved by the university’s animal care committee. MRI: Experiments were performed on a 7T Bruker Avance III NMR system with Paravision 5. Mice were anesthetized using 1.5% isoflurane in O2/N2O. Respiration and external body temperature were monitored during imaging. 4 control (CTL) and 4 cuprizone-fed (CPZ) mice underwent in vivo T2w imaging and MTI at 2, 3, 4, 5 and 6 weeks after start of treatment. After sacrifice, additional high-resolution T2w, DTI and quantitative MTI datasets were acquired. Coronal slices were selected in each mouse perpendicular to a region of the CC immediately in front of the anterior commissure. In vivo T2w and MT images were aligned using manual and mutual information image registration. Regions of interest representing both medial and lateral regions of the CC as well as the cerebral cortex (CX) were selected in the T2w images and applied to analysis of all MR methods. All image analysis was performed using MATLAB. In vivo T2w: Rapid Acquisition with Refocused Echoes (RARE – Fast Spin Echo), 3 slices, 12 averages, (2.5 cm) FOV, 98x98x750 μm resolution, effective TE 80ms, TR 1640ms, RARE factor 8, 10 minutes. In vivo MTI: Fast Low Angle Shot (FLASH – Gradient Echo Sequence), 3 slices, 48 averages, (2.5 cm) FOV, 98x98x750 μm resolution, TE 6 ms, TR 70 ms, 10° flip angle. Two images were acquired: a magnetization transfer contrast (MTC) image with MT saturation pulse (Gaussian, 10.25ms, 10μT, 6000Hz off-resonance) and a proton density (PD) image without MT saturation pulse, 2x14 minutes. Ex vivo MTI: Same pulse sequence as in vivo. 1 PD image + 24 MTC images acquired with irradiation powers of 5, 10, and 20μT and frequency offsets at each power of 100, 300, 1000, 2000, 4000, 6000, 10000, and 30000 Hz. Ex vivo DTI: Pulse Gradient Spin Echo (PGSE), tetra-orthogonal gradient-encoding scheme (7-directions), b-value = 1000 s/mm (δ = 6 ms, Δ = 14 ms), 1 slice, 6 averages, (1.25 cm) FOV, 98x98x750 μm resolution, TE 26ms, TR 5000ms, 2.5 hours. Ex vivo T2w: RARE, 1 slice, 36 averages, (1.25 cm) FOV, 49x49x750 μm resolution, effective TE 80ms, TR 1640ms, RARE factor 8, 31 minutes. Histopathology: 30 μm sections were stained with Luxol Fast Blue (LFB myelin fibres appear blue, neutrophil pink, and nerve cells purple) and Periodic Acid-Schiff (PAS glycogen appears purple-magenta).