MRI acquisitions

All MRI acquisitions were performed on a Bruker 9.4T/16 magnet (Bruker BioSpin, USA), controlled by Paravision 6 software. A custom-made volume transmit-receive radiofrequency (RF) coil designed based on Bolinger et al. [38] with an internal diameter of 40 mm was used to acquire anatomical 3D PDW and T2* images. For DWI, a similar designed Bolinger [38] transmit coil with an internal diameter of 50 mm was used for RF excitation and a 20 mm planar surface receive-only RF coil (Bruker BioSpin, USA) was used for RF reception. During MRI, the anesthetized rat was placed in the supine position onto a custom-built 3D-printed animal holder with physiological monitors attached. 3D PDW sequence: 3D PDW images were acquired using a fast low angle shot (FLASH) sequence with the following parameters [39]: repetition time (TR) = 50 ms; echo time (TE) = 4 ms; flip angle (FA) = 7°; number of signal averages (NA) = 4; spatial resolution = 0.23 × 0.23 × 0.23 mm; acquisition time = 27 min. T2* sequence: immediately following the 3D PDW scan, a multi-gradient echo (MGE) sequence with 16 evenly spaced echo times was performed using the following parameters: TR/TE/FA = 60 ms/2 ~ 32 ms/15°; NA = 2; spatial resolution = 0.23 × 0.23 × 0.23 mm; acquisition time = 16 min). To prevent image aliasing artifacts a saturation pulse was applied ventral to the lower jaw in both the PDW and MGE scans. DWI sequence: A 2D, 4 shot echo-planar spin-echo DWI imaging sequence was used for calculation of the ADC using the following parameters: TR/TE = 3000 ms/21 ms; gradient duration = 4 ms; gradient separation = 10 ms; in-plane resolution = 0.3 × 0.3 mm; 11 slices; slice thickness/gap = 2 mm/1 mm; NA = 1; acquisition time = 33 min. A total of 57 b-values ranging from 20 s/mm² to 2518 s/mm² were acquired to calculate the ADC at low, mid and high b-factor [32, 40, 41] ranges. The lowest possible b-factor was 20 s/mm² including the effect of the spoiler gradients. At each b-value, diffusion gradients were applied along three orthogonal directions (read, phase and slice encoding directions). RARE sequence: following the DWI scan, a rapid acquisition with relaxation enhancement (RARE) sequence (TR/TE/FA = 2500 ms/22 ms/90°; in-plane resolution = 0.3 × 0.3 × 2 mm; 11 slices; acquisition time = 1 min), acquired at the same resolution and orientation as the DWI sequence, was performed and utilized as an anatomical template.