Chronological T2-weighted images (T2WI) of the upper cervical spinal cord enabled the progression of spinal cord compression due to ectopic calcification to be depicted in the same twy mouse (Fig. 10.2a). DTI data sets were acquired with a spin-echo sequence based on the Stejskal-Tanner diffusion preparation. Mild and moderate spinal cord compression caused the tract fibers in DTI to detour around the ectopic calcification at 6 and 15 weeks of age, whereas the tract fibers were interrupted by severe spinal cord compression at 20 weeks of age (Fig. 10.2b).

Diffusion tensor tractography images were computed using the Diffusion Toolkit and TrackVis software (Massachusetts General Hospital, MA, USA). The diffusion tensor can be represented as an ellipsoid, where a proton at the center of the voxel has an equal probability of diffusing to any point in that ellipsoid. Fiber tracking was initiated from a manually selected region of interest (ROI), from which tracking lines were propagated bidirectionally according to the principal eigenvector in each voxel. For the tractography, an ROI was placed at the C2–3 level as the epicenter ROI and at the C0–1 level as the control ROI. We determined the number of tract fibers in each specimen at the C2–3 level (epicenter site) and the C0–1 level (control/rostral site). To analyze the sequential changes in the tract fibers of the compressed spinal cords quantitatively, we determined the canal stenosis ratios and configured tract fiber (TF) ratios (Fig. 10.3a, Table 10.1) in 6-, 15-, and 20-week-old twy mice. The TF ratio did not significantly change in the 15-week-olds, but decreased sharply in the 20-week-olds (Fig. 10.3b). To examine the significance of the depicted tract fibers, we compared the tract fibers depicted by DTT with the RT-97- and SMI31-positive fibers in the 20-week-old twy mice (Fig. 10.3c, e). The tissue sections were stained with the following primary antibodies: anti-RT-97 (mouse IgG₁, 1:200, Millipore, MA, USA) for normal neurofilaments and anti-SMI31 (mouse IgG₁, 1:200, Covance) for hyperphosphorylated normal axons. There was a positive correlation between the TF ratio and the RT-97-positive area (r = 0.7865, p = 0.0449, Fig. 10.3d) and SMI31-positive area (r = 0.7746, p = 0.0489, Fig. 10.3f).

The canal stenosis progressed with the age of the twy mice. To examine the chronological relationship between the TF ratio and canal stenosis ratio, we analyzed their correlation in five twy mice at 6, 15, and 20 weeks of age (Fig. 10.4a). As the twy mice became older, the canal stenosis became more prominent, causing a decrease in the TF ratio. Interestingly, two-part linear regression analysis [10] revealed that canal stenosis over 50 % caused a sharp decrease in the TF ratio (intersection point: 52 %, Fig. 10.4b).

Since twy mice show progressive paralysis due to the chronic spinal cord compression, we examined the motor function in the mice by the Rota-Rod treadmill test (Muromachi Kikai Co., Ltd., Tokyo, Japan) and DigiGait analysis (Mouse Specifics, Quincy, MA, USA) and compared the results with the TF ratio and canal stenosis ratio in the 20-week-old twy mice. Both the Rota-Rod treadmill latency and stride length sharply decreased when the TF ratio was below 0.9 (Fig. 10.5a, b) and canal stenosis ratio was over 60 % (Fig. 10.5c, d). Consistent with the Rota-Rod treadmill analysis, the DigiGait analysis revealed that two twy mice with severe spinal cord compression (canal stenosis ratio >60 %) could not walk on the treadmill, whereas the other twy mice with mild or moderate spinal cord compression (canal stenosis ratio <50 %) could walk on the treadmill at a speed of 8 cm/s.