Animals
Male Bio-Breeding Zucker diabetic rats (BBZDR/Wor rats) (n=8) and age-matched non-diabetic BBDR littermates (n=8), were obtained from Biomere (Worcester, MA). The obese male BBZDR/Wor rat spontaneously develops type 2 diabetes at approximately 10 weeks of age (~100%) when fed standard rat chow. BBZDR/Wor diabetic rat displays all clinical symptoms typically associated with type 2 diabetes including dyslipidemia, hyperglycemia, insulin resistance, and hypertension (7),
Rats were maintained on a 12 h:12 h light-dark cycle with a light on at 07:00 h, allowed access to food and water ad libitum and were treated with ip injections of saline at indications of weight loss. All animal experiments were conducted in accordance with the Northeastern University Division of Laboratory Animal Medicine and Institutional Animal Care and Use Committee. (https://academic.oup.com/ilarjournal/article/45/3/292/704910)
Access to rats was dependent upon the breeding schedule and resulting genotypes. This required we run two separate imaging studies, each with four rats from each genotype, separated by six months.
Imaging
Studies were done on a Bruker Biospec 7.0T/20-cm USR horizontal magnet (Bruker, Billerica, MA, USA) and a 20-G/cm magnetic field gradient insert (ID = 12 cm) capable of a 120-μs rise time. Radio frequency signals were sent and received with a quadrature volume coil built into the rat restrainer (Animal Imaging Research, Holden, Massachusetts). All rats imaged under 1-2% isoflurane while keeping a respiratory rate of 40-50 breadths/min. At the beginning of each imaging session, a high-resolution anatomical data set was collected using the RARE pulse sequence with following parameters, 35 slice of 0.7 mm thickness; field of view 3 cm; 256 × 256; repetition time [TR] 3900 msec; effective echo time [TE] 48 msec; number of excitations 3; 6 min 14 sec acquisition time.
Rats were imaged prior to and following an i.v. bolus of 6 mg/ml Fe of Ferumoxytol. The injected volume was tailored for each rat (assuming 7% blood by body weight) to produce a starting blood concentration of 200 μg/ml Fe (2× the clinical dose approved for use in humans). The QUTE-CE MRI image parameters of TE=13 µs, TR=4 ms, and flip angle =20° utilized a high radio frequency pulse pulse bandwidth of 200kHz. Therefore, the pulse duration was short (6.4µs) compared to the T2 of the approximate ferumoxytol concentration (4.58ms for 3.58mM, i.e. 200µg/ml to minimize signal blur and reduce the probability for a curved trajectory of the magnetization vector Mz. A 3x3x3 cm3 field-of-view was used with a matrix mesh size of 180x180x180 to produce 167 µm isotropic resolution.
Images were motion-corrected, aligned spatially, and resliced using MATLAB SPM12 toolbox developed at UCL (http://www.fil.ion.ucl.ac.uk/spm/). The pre-contrast UTE images were set as the baseline. For each rat in each imaging session, the voxel wise percentage change of signal intensity for each scan time point (post-con) was calculated as (post-con – baseline)/(blood intensity change) *100% as described in our previous work (10), where blood intensity change is a normalization factor calculated by the post-con blood signal intensity minus baseline blood signal intensity. A 173-region rat brain atlas (Ekam Solutions LLC, Boston, MA, US) was fit to T2-weighted RARE anatomical data set for each rat data set taken at each imaging session, using software developed at Northeastern University Center for Translational Neuroimaging (CTNI), considering the variations in brain size and positions. The fitted atlas was transferred to UTE imaging. Once the images were co-registered to the atlas, custom MATLAB code was used to mask individual brain regions for ferumoxytol measurement.
Mode of percentage change distribution for each of the 173 brain areas for control and BBZDR/Wor rats was statistically compared using a Wilcoxon rank-sum test with the alpha set at 0.05. Data was analyzed by co-authors Cai and Kulkarni blind to the identity of the groups.