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Patients: This study had institutional review board approval, and written informed consent was obtained from all subjects. Thirty-two participants were recruited as part of the Canadian ALS Neuroimaging Consortium (CALSNIC), including 16 ALS patients and 16 healthy controls. Among ALS patients, six subjects were diagnosed as possible ALS, eight were probable, and two were definite, according to the revised El Escorial criteria.
MRI: Brain MR imaging was performed on a 3T Siemens Prisma using a 64-channel receive head coil. Amide proton transfer imaging, diffusion tensor imaging, routine T1- and T2-weighted image, as well as the flip angle map were obtained. The APT sequence was modified from a standard gradient echo sequence, by adding a series of specific pre-saturation pulses at the beginning of the sequence to enable APT. Saturation power was 0.8 μT. Total saturation time was 3000 ms with duty cycle = ~94%. Other parameters included: TR = 5000 ms; TE = 1.31 ms; centric phase encoding; slice thickness = 6 mm; number of averages =1; FOV = 192 × 192 mm2; matrix = 128 × 128. Parameters for DTI were: TR = 8000 ms; TE = 60 ms; slice thickness = 2 mm (no gap); number of averages =1; matrix = 128 × 128; b1 = 1000 s/mm2, b2 = 2000 s/mm2; diffusion-encoding gradients applied in 30 noncollinear directions.
Processing: All image data was processed in Matlab using in-house software. The Z-spectrum was plotted by normalizing the different offset images to the thermal-equilibrium image. A B0 map was generated and calibrated by the Water Saturation Shift Referencing (WASSR) technique6. For diffusion, Fractional Anisotropy (FA) and Apparent Diffusion Coefficient (ADC) maps were generated by the default processing in the scanner.
Statistical analysis was performed on SPSS 24.0 (IBM, Armonk, NY). The normal distribution test was performed on each parameter first, and all parameters were confirmed to be normality. Then, analysis of covariance (ANCOVA) was used to compare APT, FA, and ADC between patients and healthy controls, and in different regions within ALS patients. The results were reported as mean ± standard deviation and p values less than 0.05 were considered statistically significant.
Within ALS patients, the amide peak was significantly different between the motor cortex and other grey matter territories (Fig. 1). Compared with healthy controls, the APT signal intensities in ALS were significantly reduced in motor cortex (P < 0.001) and corticospinal tract (P = 0.046), which was undetectable under routine imaging methods (Fig. 2). There were no statistical differences in temporal cortex (P = 0.449) and medulla (P = 0.342) between patients and controls in APT values. Although the actual type of amide losses caused by neuron death remains to be determined, previous studies supposed that it may be mediated by specific signaling pathways such as programmed cell death.
Compared with the healthy control group, fractional anisotropy (FA) values were reduced in both the corticospinal tract (P = 0.024) and temporal white matter (P = 0.001) in ALS patients. Apparent diffusion coefficient (ADC) was increased in motor cortex (P = 0.008), and the corticospinal tract (P = 0.013) in ALS patients (Fig. 3). The cerebral pathologic hallmark of ALS is the loss of upper motor neurons in the motor cortex, axonal degeneration of the corticospinal tract and lower motor neurons in the brain stem7. In agreement with pathologic findings and previous DTI studies, the decrease of FA and increase of ADC were found in the corticospinal tract in ALS patients1,8. In addition, APT was negatively correlated with FA (r = -0.477, P = 0.006) and positively correlated with ADC (r=0.629 and P < 0.001).
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