ARAS fiber tracts were identified using regions of interest (ROIs) that were manually traced on the diffusion images using TrackVis (version 5.1), an interactive image analysis software program that is available to the scientific community without charge (34 ). For Case 1, each diffusion image was compared to its corresponding histological section to ensure that radiologic ROIs shared the same borders, size, and contours as the histological ROIs. Histological ROIs were delineated by visual inspection with a light microscope and were confirmed by standard atlases of human neuroanatomy (35 –37 (link)). ROIs were identified for all of the key ARAS source nuclei implicated in arousal: cuneiform/subcuneiform nucleus, pontis oralis, median and dorsal raphe, locus coeruleus, pedunculopontine nucleus, parabrachial complex (combined medial and lateral parabrachial nuclei), and ventral tegmental area (Fig. 1A, B; Figure, Supplemental Digital Content 2, http://links.lww.com/NEN/A336 and Figure, Supplement Digital Content 3, http://links.lww.com/NEN/A337). ROIs were also traced using histological guidance for the thalamic nuclei implicated in the modulation, or gating, of arousal: the reticular nucleus, the central lateral nucleus, and the centromedian/parafascicular nuclear complex (Figure, Supplemental Digital Content 2, http://links.lww.com/NEN/A336). Each brainstem and thalamic ROI served as a seed point from which fiber tracts were generated. Because the brainstem ROIs are known to change in shape, size and contour along the rostro-caudal axis, histological guidance of ROI tracing was performed for every axial diffusion image using its corresponding histological section. Similarly, each thalamic nucleus was traced on the coronal diffusion images with direct guidance by the location of the stained nuclei on corresponding coronal tissue sections. For the intrathalamic connectivity analyses, a 2-ROI-tractography technique was utilized, based on methods developed by Catani et al (38 (link)). Specifically, fiber tracts passing between the reticular nucleus and central lateral nucleus were “virtually dissected” from fiber tracts passing between the reticular nucleus and the centromedian/parafascicular nuclear complex. For Cases 2 and 3, brainstem and thalamic nuclei were traced in accordance with the aforementioned neuroanatomic atlases. We also compared the neuroanatomic localization, contours, and boundaries of these ROIs in Cases 1, 2, and 3 to ensure consistency in the tractography analyses.
There are variations in nomenclature pertaining to the source nuclei of the ARAS in standard neuroanatomic atlases of the human brainstem. In this study, the neuroanatomic localization of the pedunculopontine nucleus was traced according to the brainstem atlas of Paxinos and Huang (35 ), extending from the caudal midbrain to the caudal border of the red nuclei. The neuroanatomic localization of the parabrachial complex was traced according to the brainstem atlas of Olszewski and Baxter (36 ), extending from the mid-pons to the rostral pons.