The experiments were performed on a clinical 3T Signa MR scanner (GE Healthcare, Waukesha, WI) equipped with a high-performance insert gradient coil (peak strength = 600 mT/m; peak slew rate = 3200 T/m/s) (14). The gradient system was operated at maximum amplitude of 500 mT/m with a slew rate of 1865 mT/m/ms to minimize vibration and acoustic problems. A custom-made quadrature proton birdcage coil (inner diameter: 44 mm) was used for both radiofrequency (RF) excitation and signal reception in phantom experiments measuring the k-space trajectories. A dual-tuned (1H/13C) quadrature coil (inner diameter: 50 mm, length: 70 mm), operating at 127.7 and 32.1 MHz, respectively, was used for the in vivo experiments. The coil was based on a previously published design (15), but with a second half-Helmholtz unit added to provide quadrature operation in the proton mode.
Polarization Procedure and Animal Handling
Substrate, polarization procedure using a HyperSense system (Oxford Instruments Molecular Biotools, Oxford, UK), and handling of the healthy male Wistar rats (168–246 g body weight) were the same as described in (5) unless stated otherwise. The solvent solution consisted of 40 mM Tris, 80 mM NaOH, 100 mg/L Disodium EDTA, and 50 mM NaCl. The final 80-mM [1-13C]-pyruvate solution had a pH of 7.4 and the liquid-state polarization was ∼20% at dissolution. Targeting a dose of 1 μmol/g, 2.1–3.0 mL were injected manually through a tail vein catheter at a rate of ∼0.2 mL/s, followed after a 1–2 s delay by a flush of 0.5 mL saline with 1% heparin. The time delay between dissolution and start of injection ranged from 19 to 29 s. All procedures were approved by the Institutional Animal Care and Use Committees at Stanford University and SRI International.
For the in vivo experiments, single-shot fast spin-echo (FSE) proton MR images in axial, sagittal, and coronal orientations were acquired as anatomical references for prescribing the 13C-spCSI experiments. In each direction, up to 45 2-mm slices were acquired with 0-mm separation and a nominal in-plane resolution of 0.47 mm (256 × 192 matrix, echo time (TE)/repetition time (TR) = 39/1492 ms). Additionally, dual-echo FSE images (0.25-mm in-plane resolution, 256 × 192 matrix, 15 1-mm slices, echo train length = 8, and TE1/TE2/TR = 11/57/5000 ms) were acquired in axial (animal coronal) direction matching the prescription in the spCSI experiments. The B0 homogeneity over the brain was manually optimized with a point-resolved spectroscopy sequence by minimizing the line width of the unsuppressed water signal with the linear shim currents.
The transmit 13C RF power was calibrated using a reference phantom containing a 8-M solution of 13C-urea in 80:20 w/w water:glycerol and 3-μL/mL Gd-chelate (OmniScan™, GE Healthcare, Oslo), which was placed on top of the animal. With a volume of only 0.8 mL for the urea solution, the effect of the phantom on the coil loading and, hence, the pulse calibration is negligible. Based on the urea signal, the center frequency was set at ∼177 ppm. The phantom was also used as a concentration reference to normalize the metabolic images. It was removed before the first spCSI measurement, because the aliased urea resonance at 163.5 ppm would partially overlap with the pyruvate (Pyr) signal. Although the phantom was next to the animal, quantitation of pyruvate would have been affected due to the distorted point spread function of urea (aliased once compared with Pyr).
In the first set of experiments, single-shot spCSI was applied to dynamic metabolic imaging of the rat brain. The spiral gradient waveforms were generated using the design algorithm of Glover (16) for a targeted field-of-view (FOV) of 43.5 × 43.5 mm2 with a 16 × 16 matrix corresponding to a nominal in-plane resolution of ∼2.7 mm. Including the refocusing lobe that returns the trajectory to the center of k-space, the duration of each spiral gradient lobe was 3.568 ms leading to a spectral width of 280 Hz. Therefore, the signals from alanine aminotransferase (Ala) and pyruvate hydrate (Pyh) were within the spectral width, whereas the signals from Pyr and lactate (Lac) were aliased once, and the bicarbonate (Bic) signal was aliased twice. The TE was 3 ms, and, with 32 lobes, the total acquisition time per image was 125 ms. A 10-mm axial (animal coronal) slice through the front and middle of the rat brain was excited with the slice center ∼1.2-mm anterior of the bregma according to the atlas of Paxinos and Watson (17). Sixteen data sets were acquired at 3-s intervals starting 9 s after the start of injection. A variable-flip-angle scheme (18) was applied for the 16 excitations to preserve the longitudinal magnetization. Although a higher temporal sampling rate of up to eight samples per second would have been possible for the single-shot acquisition, the applied sampling scheme was chosen as a compromise between the number of samples and the SNR for each sample. Each measurement was repeated three times per animal (three animals) to increase SNR and assess reproducibility. The interval between injections was ∼90 min.
In the second set of experiments, high-resolution metabolic imaging of the rat brain was performed to reduce partial volume effects using three-shot spCSI with a targeted FOV of 48 × 48 mm2 and a matrix size of 32 × 32. Number of echoes and SW were the same as in the dynamic imaging experiments. The slice location was also the same but with the thickness reduced to 5 mm. Given the lower signal due to the smaller voxel size, the excitation flip angles for the three interleaves were 35°, 45°, and 90°, respectively, to use all the longitudinal magnetization. With a TE of 3.2 ms, the total acquisition time per image was 375 ms, and the data from four injections into a single animal were acquired under the same conditions and then averaged.
Currently, the gradient driver parameters of the system have not been optimally calibrated to the impedance of the insert gradient, which leads to deviations of the k-space trajectory from the targeted design. Therefore, the trajectories for spCSI were measured using the Fourier transform method suggested by Alley et al. (19). The measurements were performed on a spherical water phantom (38-mm inner diameter) to increase the SNR. Only the trajectory for one spiral lobe was measured, and trajectories along the x and y-axes were measured separately. For each gradient waveform, two data sets were acquired with the waveform inverted for the second acquisition. Measurements were performed for a FOV of 50 mm and 256 phase encoding steps.
Data Processing and Analysis
All data processing was performed using custom software written in Matlab (MathWorks Inc., Natick, MA). For k-space trajectory measurements, the data were processed as described in (19) with the trajectories calculated from the phase of the data after fast Fourier transform. The corresponding gradient waveforms were obtained by differentiating the measured k-space trajectories.
Unless stated otherwise, the spCSI data were processed using the spectral tomosynthesis reconstruction as described in (5) with the measured k-space trajectories that were corrected for the different gyromagnetic ratios of proton and 13C nucleus. The data were apodized in the time dimension with a 10-Hz Gaussian line broadening. For three-shot spCSI, the reconstruction included an additional step correcting for phase inconsistencies of the data acquired with the three spatial interleaves (20). Metabolic images were calculated by peak integration in absorption mode with integration intervals of 36 Hz for Pyr, Lac, and Ala. The integration window for Bic was decreased to 18 Hz to reduce contributions from the nearby Pyr peak, which was only separated by ∼40 Hz due to the spectral aliasing. No metabolic images of Pyh were calculated because of its partial overlap with Pyr due to the applied spectral undersampling scheme. However, the partial overlap results in only minor contributions of Pyh to the metabolic images calculated for Pyr because of their small ratio (<8%) and the blurred point spread function of Pyh (13). The metabolic images were normalized to the signal intensity of the 8-M urea phantom expressed in institutional units (i.u.). In this notation, the urea phantom at thermal equilibrium (polarization of 2.64 × 10−4%) has an intensity of 2.1 i.u. No correction for flip-angle deviations due to B1 inhomogeneity was applied to the data. However, the animals were positioned with the brain approximately at the center of the RF coil to minimize potential flip-angle variations. Individual data sets were normalized to a liquid-state polarization of 22% and differences in transfer time from dissolution to start of injection were corrected using a T1 for Pyr of 60 s, which was measured in independent phantom experiments.