Quantifying Substantia Nigra and Locus Coeruleus Volumes

The 3D Slicer software package (version 4.11)¹ was used to measure the SN and LC. The SN was visible for four consecutive slices below the cross-section of the inferior colliculus. Similar to previous studies (Langley et al., 2015 (link); Isaias et al., 2016 (link); Wang et al., 2018 (link); Li et al., 2019 (link)), the level tracing segment editor module was used to create regions of interest (ROIs) semiautomated interactively in a blind manner by a neuroradiologist with 9 years of experience (SW). The volume and surface area of SN and LC were then calculated. ROIs for background were defined as circular areas (4 mm in diameter) in the cerebral peduncle (CP) on the left and right sides (Figure 1). The contrast-to-noise ratio (CNR) between the SN and CP was then calculated with the following equation: CNR_SN = (SI_SN–SI_CP)/SD_CP. SI_SN and SI_CP represented the mean signal intensity in the ROIs for SN and CP, respectively, and SD_CP represented the standard deviation of the ROI for CP. The values of SI_CP and SD_CP used in the equation were the average values of both CP sides.
The location of LC was identified as the spot with the highest intensity adjacent to the fourth ventricle on the bilateral sides, which was visible for three axial slices at the level of the pons. Similar to the SN assessment, the volume and surface area of LC were assessed semiautomatically. Background reference ROIs (circles with a 6 mm in diameter) were placed in the pontine (PT) tegmentum. We calculated the CNR between the LC and PT according to the following equation: CNR_LC = (SI_LC–SI_PT)/SD_PT. SI_LC and SI_PT represent the mean signal intensity in the ROIs for LC and PT, respectively, and SD_PT represents the standard deviation of the ROI for PT.