Amira 6

The nuclei of neurons were tracked manually over the course of the movie by using Slidebook 6 (Intelligent Imaging Innovations) and a Marianas Spinning Disk confocal microscope. The distance from the edge (DFE) of the slice was computed using Amira 6.2 (FEI, Thermo Fisher Scientific) and added as an intensity channel to the respective movie. Only movies in which the overall XY drift was limited to 5 μm were considered in the analysis. Nuclear coordinates (X, Y, DFE) tracked at each time-point were exported to Excel (Microsoft) in order to plot the distance distribution for each time-point and calculate the instant speed average and average distance variation. Angles were calculated from the distance variation and instant speed vectors at each time-point. Data were divided into layers based on extrapolated measurements from histologic sections at P7, i.e., oEGL: 0–30 μm; iEGL: 30–50 μm; ML: 50–100 μm; and IGL: >100 μm, based on their DFE at a given time-point.

The CT images were segmented using Amira 6 (Thermo Fisher Scientific, Waltham, MA) to create three-dimensional (3D) anatomical vertebral bone models of the cervical spine (C2-T1), as shown in Fig. 2a. These 3D vertebral bones were imported into Rhinoceros 5.0 (Robert McNeel & Associates, Seattle, WA) to establish local coordinate systems (CSs) and disc location landmarks on the vertebrae. To measure the intervertebral relative rotations and translations, two local endplate CSs were created at the centers of the proximal and distal endplate profiles in the sagittal plane, respectively (Fig. 2b); the proximal axis of each endplate CS was perpendicular to a flat plane fitting to the corresponding disc footprint surface (Fig. 2c) [2 (link),16 (link)]. In this study, the overall cervical spine motions were defined as the motions of C2 relative to T1 measured using their CSs (marked as red in Fig. 2a). To measure regional disc deformation, five points (i.e., anterior, center, posterior, left, and right) were selected on the proximal disc footprint surface (Fig. 2c); their shortest distances to the distal disc footprint surface were defined as the local disc heights corresponding to the respective five anatomic sites [18 (link),19 (link)].