Gd dota

The anesthetized mice were positioned in a stereotaxic frame and their neck slightly flexed with care to not compromise the airway and spontaneous breathing pattern. Through a midline incision, the atlanto-occipital membrane was exposed and a 34-ga shortened needle (Hamilton, US) connected via polyurethane tubing to a 50 µl Hamilton syringe (Hamilton, US) mounted in a micro-infusion pump (Legato 130, KD Scientific, Holliston, MA, USA) was inserted into the CM. Previous studies reporting on tracer delivery for GS transport studies in mice via the CM have used infusion rates of 0.1–2 µl/min for a total volume of 5–10 µl^{22 (link),37 (link)–40 (link)}. In this study we infused 7 µl of Gd-DOTA (Guerbet LLC, Princeton, NJ, US) prepared as a 1:20 dilution in sterile 0.9% NaCl or 7 µl of sterile 0.9% NaCl at an infusion rate of 1 µl/min. After the infusion, the needle was left in place for 2-min, after which it was removed, and the dura sealed with cyanoacrylate glue. Collectively, anesthesia, CM exposure and Gd-DOTA infusion required ~ 30 min.

2D MRR data were taken from five consecutive subjects enrolled in an ongoing study into MRI biomarkers of renal fibrosis. The study was approved by the local research ethics committee, and all subjects gave written informed consent (Newcastle and North Tyneside 1 Research Ethics Committee, REC reference 14/NE/1120). MRR was performed in free-breathing using a 3.0 T scanner (Philips Achieva, Best, The Netherlands) and a 2D saturation-recovery turbo-flash sequence with linear encoding of k-space. A 2-channel body transmit coil was employed for homogeneous signal transmission and data were acquired using an 18-channel torso receive coil. Four slices (3 coronal, 1 axial) were acquired at a temporal resolution of 1.1 s. Other imaging parameters were as follows: acquisition matrix 116 (phase) × 135 (read), number of dynamics 250, echo time 1.63 ms, repetition time 3.6 ms, bandwidth 900 Hz, saturation recovery time 148 ms, flip angle 12^◦, slice thickness 7 mm, sensitivity encoding (SENSE) factor 2.4, field of view 375 × 440 mm², in-plane resolution 3.2 × 3.2 mm², reconstructed matrix 480 × 480. The contrast agent Gd-DOTA (Dotarem, Guerbet, France) was injected with a half dosage of 0.05 mmol/kg body weight. The input $c_a(t)$ was measured in the aorta on the axial slice.

After routine structural MRI acquisition, DCE-MRI of the nasopharynx and upper neck was performed on a 3.0-T MRI system (Achieva; Philips Healthcare). Four acquisitions were obtained in a chronological order with a field of view (FOV) of 22 × 22 × 6 cm (AP × RL × FH): precontrast T₁-weighted fast field echo (FFE) acquisition using a flip angle of 5° (“FA5” acquisition) in 1 minute 22 seconds; T₂-weighted imaging (“T2W” acquisition) in 50 seconds; B1 mapping measurement acquisition (“B1MAP” acquisition) in 1 minute 23 seconds; and DCE acquisition using a flip angle of 15° (“FA15” acquisition) in 6 minutes 47 seconds with 65 dynamic scans. The details of scanning protocols have been described in our previous paper [9 (link)]. All four acquisitions were performed in the same anatomical region and reconstructed to the same resolution. The contrast agent Gd-DOTA (Dotarem, Guerbet, France) was injected intravenously as a bolus into the blood at around the 8th dynamic acquisition using a power injector system (Spectris Solaris, MedRad, USA), immediately followed by a 25-mL saline flush at a rate of 3.5 mL per second. The dose of Gd-DOTA was 0.1 mmol/(kg body weight) for each patient.

Brains were fixed before harvesting from animals by transcardiac perfusion of a paraformaldehyde solution 4% in phosphate buffered saline, to which 6.25 mM of Gd-DOTA was added (Guerbet Laboratories) as contrast agent. The contrast agent was used here to accelerate MRI acquisition time. After removing surrounding skin and muscles, the skulls containing intact brains were immersed in the fixing solution for four days, and then transferred to a Fomblin (FenS chemicals) bath for at least eleven days after brain fixation. This schedule provided homogeneous distribution of the Gd-DOTA throughout the whole brain^{31 (link)}.

Skulls were processed as described in Pagnamenta et al., 2019 (link). Briefly, four male mice of each genotype aged between 8 and 11 weeks were transcardially perfused with 4% paraformaldehyde solution in phosphate buffered saline containing 6.25 mm of Gd-DOTA (Guerbet Laboratories, Roissy, France). This contrast agent is added to reduce the MRI acquisition time. Skin and head muscles were removed to expose the skull, which was then immersed in the fixing solution for 4 days. The skull was then transferred to a Fomblin (FenS chemicals, Goes, Netherlands) bath for at least 7 days for the distribution of Gd-DOTA to be homogeneous throughout the whole brain.