Gadolinium-enhanced Ex Vivo MRI of Mouse Brains

Active stained gadolinium-enhanced ex vivo T2* MRI scans of the mouse brains were performed at age 18–21 weeks. Mice were sacrificed with an overdose injection of sodium pentobarbitone. An initial saline flush (1520 ml) was administered to the left ventricle, followed by a perfuse-fixation with 50 ml of 4% buffered formal-saline (Pioneer Research Chemicals, Colchester, UK) with 8 mM Gd-DTPA (Magnevist, Bayer-Schering Pharma, Newbury, UK), both at a flow rate of 3 ml/min. The decapitated intact skulls were then post-fixed in a solution of 4% formal saline and 8 mM Gd-GTPA at 4 °C for 9 weeks.
The ex vivo images were acquired following the protocol introduced by Cleary et al. (J.O. Cleary et al., 2011 (link)) to optimize the tissue contrast between different neuronal layers. The in-skull brains were imaged on a Varian 9.4T DirectDrive VNMRS system (Varian Inc., Palo Alto CA, USA) with a 26 mm quadrature volume coil (RAPID Biomedical GmbH, Wrzburg, Germany). 3D spoiled gradient echo sequence was used, with the following scanning parameters: TE = 4.03 ms, TR = 17 ms, FA = 52°, FOV = 20.48 × 13.04 × 13.04mm³, matrix = 512 × 326 × 326, averages = 6, scan time = 3 h. In addition, we used a multi-brain scanning protocol to image three brains at once to achieve multi-brain separation, orientation correction, and brain mask extraction (Powell et al., 2016 (link)).