Clinical Need for DTT of the Spinal Cord: The spinal cord white matter is composed of many nerve fiber bundles, but tissue specimens appear uniform, and it is impossible to observe the individual projections unless a special dye is used. Moreover, because proton relaxation times are very similar for different fiber bundles, it is impossible to tell them apart by DTI, even using the black and white contrast provided by T1- or T2-weighted MR imaging (T1WI or T2WI). Thus, although information about fiber tracts and the integrity of axons in the spinal cord is extremely important, it has been difficult to obtain in vivo. Even when the latest artifact-reducing imaging sequences and high-magnetic-field MRI scanners are used, the fiber bundles comprising individual tracts in the spinal cord white matter cannot be distinguished. To make a prognosis or select the appropriate treatments for the spinal cord injuries that we have discussed here, it is extremely important to know where axons have been destroyed or demyelinated and to what extent axons have been spared. Until now however, even using the best imaging technology available, T1WI and T2WI, only the location of the injury and the degree of compression have been detectable.

In the last decade or so, many reports have described the use of DTT to depict neuronal fibers in the white matter of the brain. High-resolution data for the spinal cord, in contrast, has been difficult to obtain, because the spinal cord is smaller than the brain, it is located deep in the body, and imaging it requires discriminating among tissues and materials with different magnetic properties, such as the spinal cord white matter, cerebrospinal fluid, vertebrae, muscle, and air that are intermingled within a small space [19, 20]. Thus, although occasional reports on the use of DTT to depict the spinal cord in humans have been published, their precision has been low, and few have included histological studies [23–25]. To apply DTT of the spinal cord clinically, this method must be verified by conducting a detailed comparison between the DTT and histological findings. We therefore performed DTT imaging of the spinal cord in a primate, the common marmoset, and examined its accuracy by conducting comparative tissue studies.

DTT of the Cervical Cord of a Primate, the Common Marmoset: We created a reproducible model of SCI in the common marmoset, which is a primate and therefore more closely related than rodents to humans, and found that the transplantation of human neural stem cells contributed to functional recovery in this model [1, 8]. Unfortunately, there are still no reports of curing complete spinal cord injuries in humans, although functional recovery can be anticipated if approximately 5–10 % of the axons are spared from injury. At the animal experiment level, injured axons can be evaluated histologically by injecting a tracer, such as biotinylated dextran amine (BDA), into the primary motor cortex of the cerebrum, but such an analysis obviously cannot be used in the clinic.

To examine whether DTT might be used to assess injured axons in humans, we first used it to examine common marmosets with spinal cord injuries. In these animal experiments, the images and spin-echo sequence were acquired by a 7-Tesla MRI, and we obtained the first clear DTT depiction of the primate spinal cord [37]. To determine whether the DTT results accurately reflected the condition of the axons, we created a simpler cervical cord (C5/6) hemisection injury model and performed DTT 2 weeks later after sacrificing the animal (postmortem model). In a reconstruction of the nerve fiber interruption, DTT clearly depicted the hemisection injury, which was detectable by conventional MRI only in the form of T1 and T2 contrast (Fig. 28.4). Moreover, the DTI and DTT results reflected the histological results obtained with Luxol fast blue (LFB) and other staining methods.

Next, by optimizing the depth of anesthesia to minimize artifacts from respiratory movements and cerebrospinal fluid pulsation, and by making various modifications based on the findings in the postmortem model, we were able to use DTT to reproducibly depict the different fiber tracts of the spinal cord in living common marmosets (Fig. 28.5). Moreover, we were able to depict the pyramidal decussation with DTT, which has been difficult to depict by other methods (Fig. 28.6), and demonstrated that DTT was could be used as a tool for fiber tracking instead of conventional tracers.

We also performed longitudinal DTT, behavioral, and histological analyses before and after contusive SCI in common marmosets. By comparing the tract fiber estimate depicted by DTT with neuronal fibers labeled with anti-neurofilament antibodies, we determined the optimal fractional anisotropy (FA) threshold for fiber tracking to be 0.40. The ratio of the number of tract fiber estimate at the lesion site to the number before SCI was significantly correlated with the functional recovery after SCI. Moreover, comparison of the longitudinal pre- and post-SCI FA and axial diffusivity (λ_∥) values revealed that they decreased after injury at the sites caudal to the lesion epicenter in the CST and rostral to the lesion epicenter in the dorsal column [39] (Figs. 28.7 and 28.8). In summary, diffusion imaging is more sensitive than conventional MRI for precisely determining the extent of spinal disorders via noninvasive, longitudinal examinations, in both human patients and animal models. Moreover, FA analysis may prove more useful than other diffusional indices, because of its simplicity, accuracy, and ability to reveal diverse spinal cord disorders especially in the clinical situation.

As the performance of MRI devices improves, the number of DTI studies of the spinal cord should increase. There are two categories of such studies. One involves visualizing the spinal cord disorders by tractography, including the visualization of inflammatory and degenerative disorders [40, 41], the localization and characterization of spinal cord tumors [25, 42], and characterization of the deformation and interruption of local fibers caused by arteriovenous malformations [43] and Brown-Sequard syndrome [44].

The other category involves the quantitative analysis and examination of correlations between diffusion and both functional and histological conditions. Budde et al. revealed that axial diffusion in the ventrolateral white matter in the experimental autoimmune encephalomyelitis (EAE) mouse model was significantly negatively correlated with the EAE clinical score and was significantly lower in mice with severe EAE than in mice with moderate EAE [45]. In the ventral white matter tracts of the spinal cord of ALS-affected mice, the diffusion values altered with disease progression [46, 47]. In an SCI study, Kim et al. identified the spared, normal ventrolateral white matter by the hyperacute axial diffusion 3 h after injury, and a histological analysis performed 14 days later showed a good correlation with the spinal contusion injury [48]. Loy et al. demonstrated that axial diffusion in ventral white matter differentiated mild, moderate, and severe contusive SCI, with good histological correlation [49]. In summary, these recent studies indicate that the quantitative DTI parameters are sensitive and specific biomarkers for spinal cord white matter integrity.

Several clinical studies suggest that the increased sensitivity of DTI makes it appropriate for the early detection of myelopathic changes in patients with cervical myelopathy. A significant positive correlation between the FA at the compressed level and the clinical assessment has been demonstrated [50, 51]. This correlation was also seen in patients with SCI [23, 52]. Chang et al. revealed that quantitative fiber tract results correlate to some extent with the severity of cervical SCI in patients [53].

Resolution: DTT does not depict the axons themselves, although this is frequently misunderstood. The voxel size required for analyzing anisotropy is far greater than the diameter of an axon, so the diffusion anisotropy of fiber bundles composed of 10²–10³ axons or more is what is actually depicted.

Depiction of Complicated Axonal Pathways: Closely related to the resolution problem, the anisotropies of fibers oriented in different directions within the same voxel sometimes cancel each other out. This is the result of a partial volume effect that stops the tracking. Conversely, if the anisotropy is not cancelled out, the sum of the conflicting information allows tracking, but sometimes leads to the depiction of a structure that cannot actually exist. Therefore, DTT images do not always reflect actual anatomical structures. We have addressed this issue by setting a threshold value for FA that reduces the likelihood of depicting an erroneous tract and setting a threshold value for anisotropy tracking that prevents sharp changes in direction. At present, we consider it necessary to interpret DTT results cautiously, drawing on all of the anatomical and histological information available, just as when reading ordinary MRI scans. Complicated, multiaxial neural pathways can be analyzed by using a high b value³ [54, 55]. By increasing the b value, we have succeeded in clearly depicting the optic chiasm (Fig. 28.9), brain stem, and cervical nerve roots (Fig. 28.10), but because these analyses require an enormous amount of time, they have not reached the point of clinical application.

Thus, the b value is an index that indicates the degree of diffusion, and higher b values mean greater diffusion. For the DTT images that we obtained in this study, we used b = 1,000. By contrast, a “high” b value is 2,500 or more, which makes it possible to detect intracellular diffusion and the diffusion of more complicated nerve pathways that cannot be perceived by conventional DWI.

By exploiting our DTT findings from the spinal cords of marmosets, we have already applied this method clinically to a fairly large number of cases. We are currently assessing the usefulness of DTT for examining cervical spondylotic myelopathy [56] (Fig. 28.11), ossification of the posterior longitudinal ligament (OPLL), and spinal cord tumors, as well as SCI [31] (Fig. 28.12). The application of DTT is restricted to diseases of the cervical region, because it is difficult to obtain high-resolution images in the trunk, which currently prevents detailed analysis. We hope that future advances in the development of MRI hardware and software will enable researchers to overcome the problems of long image acquisition time and resolution. Moreover, we believe that by revealing more anatomical structural information about the tracts, it will be possible to detect and evaluate the lesions of even more spinal cord diseases.