Mall size of the mouse and its respiratory and cardiac movements. Cowin et al used ex vivo MRI to show subtle anatomical spinal cord changes in transgenic mice [24]. In the present study, use of the CryoProbe, an innovative MRI probe, enabled high-resolution in vivo MRI of the spinal 1379592 cord in WT and CST-KO mice. Both in vivo and ex vivo MRI of the spinal cord revealed Calyculin A web Lixisenatide decreased T1 times and increased T2 times in CST-KO mice as compared to WT mice. Consistent with these findings, previous reports have indicated that decreased T1 time and increased T2 time are related to myelination disorders [25,26]. These results support the utility of in vivo MRI with the Cryoprobe to detect the subtle pathological changes present in white matter disorders. Although DTI effectively reflects histological findings, in vivo DTI is difficult to perform due to the lengthiness of the procedure; therefore, we also performed ex vivo DTI. We found low AD and high RD in the CST-KO spinal cord. Micro-histological examination detected differences in the axonal and myelinated structures between CST-KO and WT mice. Although Song et alFigure 3. Diffusion tensor imaging in the spinal cords of WT and CST-KO mice. (A) FA map and ROI analysis of the FA values. The FA value of the CST-KO mice was significantly lower than that of WT mice. (B) Axial and radial diffusivity (AD and RD) maps and ROI analyses of the diffusivities. The AD of CST-KO mice was significantly lower than that of WT mice, whereas the RD was significantly higher. (A, B) Values shown are means 6 s.d. (ex vivo: n = 7), with statistical significance determined by the unpaired t-test. ***: p,0.001, **: p,0.01, *: p,0.05. doi:10.1371/journal.pone.0052904.gand in vivo T1 times for the spinal cord were significantly lower in CST-KO mice than in WT mice (Fig. 2A). On the other hand, the ex vivo and in vivo T2 times were significantly higher in CST-KO mice than in WT mice (Fig. 2B). After generating DTI maps (e.g., FA, AD, and RD) of the spinal cord in WT and CST-KO mice, the FA values at the ventral side of the spinal cord were measured to analyze DTI differences between WT and CST-KO mice. The FA values were significantly lower in CST-KO than in WT mice (Fig. 3A). The AD values (diffusivity parallel to the long axis of the spinal cord) (Fig. 3B) were also significantly lower in CST-KO than in WT mice. The RD values (diffusivity perpendicular to the long axis ofMRI Findings of Paranodal Junction FailureFigure 4. Histological analysis of the paranodal junctions and axons in the spinal cords of WT and CST-KO mice. (A) Representative images of the axons in sagittally sectioned spinal cords stained for Na+ channel, Caspr, and K+ channel. Scale bar: 20 mm; enlarged view: 5 mm. 15826876 (B) Quantitative analysis of the number of paranodal clusters per field of view. (C) Ultrastructure of the paranodal junction at a node of Ranvier in a WT and a CST-KO mouse, shown by electron microscopy (arrowheads). Scale bar: 1 mm. (D) Representative toluidine blue-stained images of axially sectioned spinal cord axons. Focal axonal swelling was conspicuous in the white matter of the CST-KO spinal cord (arrows). Scale bar: 50 mm. (E) Quantitative analysis of the axon density. (B, E) Values show the means 6 s.d. (n = 4), and significant differences were determined by the MannWhitney test. *: p,0.05. doi:10.1371/journal.pone.0052904.greported that AD and RD are markers of axonal and myelin degeneration, respectively [9,27,28], we found that par.Mall size of the mouse and its respiratory and cardiac movements. Cowin et al used ex vivo MRI to show subtle anatomical spinal cord changes in transgenic mice [24]. In the present study, use of the CryoProbe, an innovative MRI probe, enabled high-resolution in vivo MRI of the spinal 1379592 cord in WT and CST-KO mice. Both in vivo and ex vivo MRI of the spinal cord revealed decreased T1 times and increased T2 times in CST-KO mice as compared to WT mice. Consistent with these findings, previous reports have indicated that decreased T1 time and increased T2 time are related to myelination disorders [25,26]. These results support the utility of in vivo MRI with the Cryoprobe to detect the subtle pathological changes present in white matter disorders. Although DTI effectively reflects histological findings, in vivo DTI is difficult to perform due to the lengthiness of the procedure; therefore, we also performed ex vivo DTI. We found low AD and high RD in the CST-KO spinal cord. Micro-histological examination detected differences in the axonal and myelinated structures between CST-KO and WT mice. Although Song et alFigure 3. Diffusion tensor imaging in the spinal cords of WT and CST-KO mice. (A) FA map and ROI analysis of the FA values. The FA value of the CST-KO mice was significantly lower than that of WT mice. (B) Axial and radial diffusivity (AD and RD) maps and ROI analyses of the diffusivities. The AD of CST-KO mice was significantly lower than that of WT mice, whereas the RD was significantly higher. (A, B) Values shown are means 6 s.d. (ex vivo: n = 7), with statistical significance determined by the unpaired t-test. ***: p,0.001, **: p,0.01, *: p,0.05. doi:10.1371/journal.pone.0052904.gand in vivo T1 times for the spinal cord were significantly lower in CST-KO mice than in WT mice (Fig. 2A). On the other hand, the ex vivo and in vivo T2 times were significantly higher in CST-KO mice than in WT mice (Fig. 2B). After generating DTI maps (e.g., FA, AD, and RD) of the spinal cord in WT and CST-KO mice, the FA values at the ventral side of the spinal cord were measured to analyze DTI differences between WT and CST-KO mice. The FA values were significantly lower in CST-KO than in WT mice (Fig. 3A). The AD values (diffusivity parallel to the long axis of the spinal cord) (Fig. 3B) were also significantly lower in CST-KO than in WT mice. The RD values (diffusivity perpendicular to the long axis ofMRI Findings of Paranodal Junction FailureFigure 4. Histological analysis of the paranodal junctions and axons in the spinal cords of WT and CST-KO mice. (A) Representative images of the axons in sagittally sectioned spinal cords stained for Na+ channel, Caspr, and K+ channel. Scale bar: 20 mm; enlarged view: 5 mm. 15826876 (B) Quantitative analysis of the number of paranodal clusters per field of view. (C) Ultrastructure of the paranodal junction at a node of Ranvier in a WT and a CST-KO mouse, shown by electron microscopy (arrowheads). Scale bar: 1 mm. (D) Representative toluidine blue-stained images of axially sectioned spinal cord axons. Focal axonal swelling was conspicuous in the white matter of the CST-KO spinal cord (arrows). Scale bar: 50 mm. (E) Quantitative analysis of the axon density. (B, E) Values show the means 6 s.d. (n = 4), and significant differences were determined by the MannWhitney test. *: p,0.05. doi:10.1371/journal.pone.0052904.greported that AD and RD are markers of axonal and myelin degeneration, respectively [9,27,28], we found that par.