Bga 12

The volume of the right brain hemisphere, white matter, and grey matter were measured by MRI as previously described (Henriksen et al., 2022a (link)). Briefly, the entire right hemisphere was immersed in 4% paraformaldehyde in phosphate buffered saline for 24 h at 4°C (PBS; 0.1 M, pH 7.4), rehydrated with PBS for 24 h at 4°C and washed twice with Fomblin (perfluoro-polyether; Solvay, Princeton, New Jersey, USA) to remove the excess fluid and resubmerged in clean Fomblin prior to scanning. It was confirmed that there was no surrounding fluid signal on the surface of each brain before high-resolution imaging. Data was acquired with a 9.4 Tesla preclinical scanner (Bruker BioSpin, Ettlingen, Germany) equipped with a 240 mT/m gradient coil (BGA-12S, Bruker) and using an 86-mm inner diameter transmit-receive volume coil. The imaging protocol used a 3D gradient-spoiled steady state free precession, and the parameters were set to: repetition time = 4.6 ms, echo time = 2.3 ms, number of signals averaged = 10, flip angle = 25°, field of view = 60 mm × 38.4 mm × 25.6 mm, matrix = 300 × 192 × 128, image resolution = 200 μm isotropic, acquisition time = 20 min. The images were bias-field corrected, and segmented as previously described (Henriksen et al., 2022a (link)). The volume of white and grey matter was normalized to the total volume of the brain hemisphere.

To achieve high-spatial resolution of MRI CSF space volumetry and cisternography a 3D constructive interference in steady-state (CISS) sequence along with a cryogenically cooled Tx/Rx quadrature-resonator (CryoProbe, Bruker BioSpin) and 240 mT/m gradient coil (BGA-12S) were used. During MRI, the animals (5 KO and 6 WT) were anesthetized under Ketamine/Xylazine (i.p. K/X: 100/10 mg/kg) and underwent acquisition of two 3D-TrueFISP volumes of opposite phase encoding direction (i.e.: 0° and 180°) (Table 1B), for further 3D-CISS image calculation. The complete MRI protocol lasted over an hour so every animal was implanted with a permanent intraperitoneal PE-10 catheter in the abdominal area, connected to a 1 mL syringe filled with K/X solution. The syringe was kept outside MR during whole imaging, and animals received a single supplementary dose of K/X after the first TrueFISP volume acquired. No difference in age (p=0.697 for KO vs. WT using Mann-Whitney U-test), body weight (p=0.7662), respiration rate during MRI (p>0.99) as well as signal-to-noise ratio (SNR) of computed 3D-CISS images (3.5 ± 0.4 vs 3.7 ± 0.2; p=0.1385) was found between KO and WT animals (Table 1A) so no animals were excluded from further analysis.

All imaging experiments were carried out on a BioSpec 70/30 USR 7 Tesla small animal MRI system (Bruker Corporation, Ettlingen, DE) equipped with a B-GA12 gradient coil insert and 35 mm inner diameter quadrature transmit-receive cylindrical ¹H RF volume resonator. Results used to determine contrast agent sensitivity limits were acquired in triplicate.

Animals were anesthetized with 1% isoflurane in a 30:70% O₂:N₂O gas mixture and imaged in a horizontal bore 7-Tesla USR preclinical MRI system (BioSpec 70/30, Bruker BioSpin, Germany) with a shielded gradient insert (BGA 12, 400 mT/m; rise time, 110 us). A 72-mm birdcage resonator for RF transmission, and a 10-mm diameter single-loop receiver coil were used to receive the signal. 3D T2-weighted anatomical images of the mouse brain were acquired with the following parameters: TR 2500 ms, TE 50 ms, RARE factor 16, FOV 3 x 1.5 x 1.5 cm, Matrix 256 x 102 x 102, voxel 0.147 x 0.117 x 0.147. The scan time was approximately 25 min. The volumes of the whole brain and individual brain areas (frontal cortex, hippocampus, thalamus, striatum and cerebellum) were quantified manually using Fiji software [79 (link)], after a rigid body registration (6 dof) to a reference image to avoid bias due to bad head positioning.