External Capsule

Each histological assessment was performed on five sections 400 μm apart. Infarct size was measured using Optimas 6.5 image software, and the intact areas of the isocortex, pyriform cortex, hippocampus, striatum, thalamus and external capsule white matter were delineated bilaterally and measured as described previously (Kendall et al., 2012 (link)). Injury score (see Table 1) based on combined microglial activation (αM immunoreactivity) and neuronal cell loss (Nissl stain) was assessed in the same aforementioned brain regions and adapted from a scoring system previously established in our laboratory (Kendall et al., 2006 (link)). The presence of cell death involving DNA fragmentation was detected through quantification of the number of TUNEL positive nuclei in three separate fields (×20 magnification) per assessed brain region. Mean and standard deviation of optical luminosity values were measured in three fields (×20 magnification) of the different brain regions of GFAP-stained slides using Optimas 6.5 image software (Kendall et al., 2011 (link)). All assessments were performed by an observer blinded to the different treatments. Additionally, each assessment was performed for each of the strains simultaneously.

We defined the BLA as composed of basal, lateral, and accessory basal nuclei of the amygdala, as previously reported [7 (link)]. Manual ROIs were carefully drawn on the right and left BLA regions on slices encompassing the amygdala on DTI parametric maps based on known anatomical locations from atlases [32 (link)]. The amygdala complex starts at approximately Bregma −1.30 and extends to −4.80 mm. The basal and lateral nuclei (BLA) are found within this antero-posterior range from −1.40 to −3.80 mm. For our analysis, the BLA was bounded by the external capsule, a white matter tract that is readily discernible on MRI. The dorsal extent of the BLA starts at the level of the rhinal fissure and extends 1.5 to 2 mm ventrally. The BLA is also bounded by the striatal, cortical (piriform) and ventricular structures (central and medial nuclei of the amygdala) in the medial aspect. Volumetric analyses of the total brain and BLA were performed on MR coronal slices using Cheshire image processing software (Hayden Image/Processing Group, Waltham, MA, USA, RRID:SCR_018225). For the volumetric data, bilateral BLA boundaries were manually drawn on each data set from the slice-shifted directionally encoded DTI image series using the boundaries as described above. Areas from each slice were extracted and multiplied by the effective interslice distance (200 µm) and slice number to obtain BLA volumes. All data were extracted and summarized in Excel.
To further demonstrate the robustness of the described approach, we undertook inter-rater calculations of the amygdala volumes from a random subset of three animals. Rater A derived the original delineations reported herein, Rater B had limited experience with MRI or amygdala anatomy, whilst Rater C has extensive knowledge of both. Raters B and C were provided with a protocol and a coded dataset with no additional training. After initial assessments, Rater B (least experienced) was provided with additional training and knowledge of the amygdala and then retraced the volumes 60 days later. We also tested for test–retest reliability, where Rater B re-acquired all volumes 30 days after the first set of delineations. No significant differences between amygdala volumes were found (see Supplemental Figure S4).

Measurements were performed with a Biospec 70/30 spectrometer (Bruker Medical Systems, Ettlingen, Germany) operating at 7T. The operational software of the scanner was Paravision 5.1 (Bruker). Images were acquired from anesthetized, spontaneously breathing animals using a circularly polarized coil (Bruker, Model 1P T20063V3; internal diameter 23 mm) for radiofrequency excitation and detection. Neither cardiac nor respiratory triggering was applied. Following a short period of introduction in a box, mice were maintained in anesthesia with 1.5% isoflurane (Abbott, Cham, Switzerland) in oxygen, administered via a nose cone. During MRI signal acquisitions, animals were placed in prone position in a cradle made of Plexiglas, the body temperature was kept at 37 ± 1 °C using a heating pad, and the respiration was monitored.
A T₂-weighted, two-dimensional multislice RARE (Rapid Acquisition with Relaxation Enhancement) sequence [34 (link)] was used for selecting the regions-of-interest (ROIs) and for evaluating signal intensities. A two-dimensional multislice gradient-recalled FLASH (Fast Low-Angle Shot) acquisition [35 (link)] served to assess the magnetization transfer ratio (MTR), a measure reflecting myelin content (Beckmann et al., 2018). Assessments of the relaxation time T₂ were performed using a multislice spin-echo sequence. As the three sequences had the same anatomical parameters, the ROIs for evaluations were selected on the RARE images and then transferred to the FLASH and spin-echo images. MRI images were analyzed using the Paravision software.
The parameters of the acquisitions were the following: (a) RARE sequence: effective echo time (TE) 80 ms, repetition time (TR) 3280 ms, RARE factor 16, 12 averages. Hermite pulses of duration/bandwidth 1 ms/5400 Hz and 0.64 ms/5344 Hz were used for radiofrequency excitation and refocusing, respectively. Fat suppression was achieved by a gauss512 pulse of 2.61 ms/1051 Hz duration/bandwidth followed by a 2-ms-long gradient spoiler. The total acquisition time was of 7 min 52.3 s; (b) FLASH sequence: TE/TR 2.8/252.8 ms, 4 averages. A hermite pulse of 0.9 ms/6000 Hz duration/bandwidth and flipangle 30° was used for radiofrequency excitation. MTR contrast was introduced by a gauss pulse of 15 ms/182.7 Hz duration/bandwidth applied with radiofrequency peak amplitude of 7.5 μT and an irradiation offset of 2500 Hz. The acquisition was then repeated with the same parameters but without the introduction of the MTR contrast. MTR was then computed using the formula MTR = (S₀ − S_MTR)/S₀, where S₀ and S_MTR represent, respectively, the signal intensities in the FLASH acquisitions without and with the introduction of the MTR contrast. The total acquisition time for both data sets was of 6 min 31.6 s; (c) assessments of the relaxation time T₂ were performed using a multislice spin-echo sequence with the parameters: 16 echoes spaced by 11 ms, TE from 11 to 176 ms, TR 3000 ms, fat suppression as described above. T₂ was determined by exponentially fitting with Origin Pro^® 2021 (OriginLab, Northampton, MA, USA) the mean signals as function of TE from ROIs placed in the corpus callosum (cc) and the external capsule (ec). All three sequences were run using the same anatomical parameters: field-of view 20 × 18 mm², matrix size 213 × 192, pixel size 0.094 × 0.094 mm², slice thickness 0.5 mm, 15 adjacent slices.