The overall process for the hyperpolarized imaging experiments was as follows: prepare a sample to be polarized, polarize with a DNP polarizer, perform anatomical imaging of the rat while waiting for the polarization to build up, dissolve and eject a liquid sample from the polarizer, inject the sample into a rat inside a clinical magnetic resonance (MR) scanner, and perform either slice dynamic spectroscopy or 3D-MRSI. A total of 20 rat experiments were conducted (five normal dynamic, five fasted dynamic, five normal MRSI, five fasted MRSI).
Polarizer and Preparations
A HyperSense DNP polarizer (Oxford Instruments, Abingdon, UK) was used in this study. Following previously described methods [4
], 32 μL (about 40 mg) of [1-13
C]pyruvic acid (Isotec, Miamisburg, OH, USA) with 15 mM OX63 trityl radical (Oxford Instruments, Abingdon, UK) was polarized in a field of 3.35 T at approximately 1.4 K by irradiation with 94.116-GHz microwaves. After approximately 1 h when the solid-state polarization neared its exponential asymptote, an aqueous solution with 5.96 g/L Tris (40 mM), 4.00 g/L NaOH (100 mM), and 0.1 mg/L Na2
ethylenediaminetetraacetate acid was injected into the DNP polarizer, heated, and used for rapid thawing and dissolution of the solid-state sample. The amounts of solvent, NaOH, and Tris buffer used were calculated to produce a final polarized sodium pyruvate concentration of 100 mM and a pH of approximately 7.6. After the rapid thawing, the dissolved material was ejected into a flask resting on ice. Immediately thereafter, a small aliquot (~0.5 mL) of the hyperpolarized [1-13
C]pyruvate solution was used to measure the level of polarization achieved in solution, and at the same time another ~2.4 mL was taken to the MR scanner to be injected into a rat over a 12-s period followed by a normal saline flush.
Animal Handling All animal studies were carried out under a protocol approved by the University of California San Francisco Institutional Animal Care and Use Committee. Male Sprague-Dawley rats weighing ~300 g were either allowed to feed freely on standard rat chow (normal, nonfasted) or had their food removed 24 h before each hyperpolarized study (fasted). For each experiment, the rat was placed on a heated pad and anesthetized with isoflurane (2–3%). A catheter was introduced into the tail vein for the eventual intravenous administration of hyperpolarized pyruvate solution, and the rat was transferred to a heated pad in the radio frequency (RF) coil in the MR scanner. While in the scanner, anesthesia was maintained by a continual delivery of isoflurane (1–2%) via a long tube to a nose cone, with an oxygen flow of 1 L/min. The rat’s vital signs (heart rate and oxygen saturation) were continually monitored. Care was taken to ensure that body temperature was maintained at 37°C throughout the imaging procedures by maintaining a flow of heated water through the pad underneath the rat.
MRI, MRS, and MRSI Studies
All studies were performed using a 3T GE Signa™ scanner (GE Healthcare, Waukesha, WI, USA) equipped with the multinuclear spectroscopy hardware package. The RF coil used in these experiments was a dual-tuned 1
C coil with a quadrature 13
C channel and linear 1
H channel construction based on an earlier design [23
] and used in prior hyperpolarized 13
C pyruvate rat imaging studies [10
]. The inner coil diameter was 8 cm, and the length of the coil was 9 cm to accommodate rats of varying size.
T2-weighted anatomical images were obtained in all three planes using a fast spin-echo sequence. Axial and sagittal images were each acquired in approximately 10 min with a 10-cm field of view (FOV), 192
192 matrix, 2-mm-thick slices and NEX
6. Coronal images were acquired with a 12-cm FOV, 192
192 matrix, 1.5-mm-thick slices and NEX
6 with a scan time of 10 min. The total imaging time required to obtain images in all three planes was thus approximately 30 min.
For the slice localized liver dynamic MRS experiments, a double spin-echo pulse sequence with a 5° flip selective RF excitation pulse (15-mm axial slice localization) and a pair of nonlocalized 180° hyperbolic secant refocusing pulses was used [24
]. A TE of 35 ms (half-echo collected), a repetition time (TR) of 3 s, and a readout filter of 5,000 Hz/2,048 pts were used for these studies. The acquisition localized to a slice through the liver started at the beginning of a 12-s manual injection of [1-13
C]pyruvate into the rat tail vein.
The 3D 13
C MRSI data were acquired in 14 s (starting 25 s after the start of injection) with a slab-selective variable small tip angle excitation pulse, double spin-echo refocusing pulses, and a flyback echo-planar readout trajectory [11
]. An 8
8 phase encoding matrix with a flyback echo-planar trajectory on the z
16 effective matrix) was used with 10
10-mm spatial resolution (1.0-cm3
voxel resolution) with an 80
160-mm FOV to cover the rat torso and abdomen. The flyback echo-planar trajectory was designed for a 581-Hz spectral bandwidth to include 13
C lactate, 13
C alanine, and 13
C pyruvate without spectral aliasing. A total of 59 readout/rewind lobes were included during each readout for a spectral resolution of 9.83 Hz. With a readout filter of 25,000 Hz/2,538 points, 16 k-space points were acquired during each TR [25
]. The TE for the MRSI acquisition was 140 ms (readout was centered on the center of the second spin-echo, full echo collected), and the TR was 215 ms. As described previously [11
], a variable flip angle (VFA) scheme [26
], with increasing flip angle over time to compensate for the loss in hyperpolarized signal, was used in the in vivo
experiments. Reordering of phase encodes to collect data near the k-space origin first was also employed as previously described [11
Fig. shows an example of the dynamic and spectral parameters for data processing in the slice MRS and 3D-MRSI experiments. The MRS example comes from data plotted in Fig. , and the 3D-MRSI example comes from a voxel from Fig. . Dynamic MRS data, processed with MATLAB™, were apodized in the time domain with a 10-Hz Lorentzian filter, Fourier-transformed along the time dimension and taken from the resulting magnitude spectra. Magnitude dynamic curves were obtained for pyruvate, pyruvate-H2
O, lactate, and alanine, from which peak lactate to alanine ratios were derived (Fig. left). A nonparametric statistical test (used to avoid any normality assumptions), the Mann–Whitney rank-sum test, was used to compare the peak lactate to alanine ratio between normal and fasted groups. For 3D-MRSI acquisitions, the reconstruction and analysis procedures, described previously in more detail [11
], were carried out with specialized custom MRSI software [27
] as follows: (1) the raw flyback data were subsampled and ordered to obtain a 4D matrix of k-space data, (2) each FID was apodized and a linear phase correction was applied to the spectral samples as described in [25
] to account for the tilted k-space trajectory characteristic of the flyback readout, and (3) a 4D Fourier transform with zero padding of the spectra was performed. From the processed 3D magnitude spectral data, for each rat, the voxels localized to nonvasculature liver tissue were identified from the proton anatomical images and the 13
C spectra with sufficient signal-to-noise ratios (>5) were quantified. For each liver voxel, the area under the spectral resonances of pyruvate, pyruvate-H2
O, lactate, and alanine were calculated, with the sum of these four areas defined as the total carbon area (Fig. right). Lactate area to total carbon area and alanine area to total carbon area were calculated for each voxel and then averaged over all selected liver voxels to derive the test statistics average lactate to total carbon ratio and average alanine to total carbon ratio. The Mann–Whitney rank-sum test was used to compare these test statistics between normal and fasted groups.
Fig. 2 Example demonstrating the quantification procedures for slice MRS and 3D-MRSI. 13C-lactate and 13C-alanine peak heights were important parameters in the final processed dynamic curves (left). The areas of hyperpolarized 13C resonances in the 3D-MRSI spectra (more ...)
Fig. 3 Comparison of normal (top) and fasted (bottom) rat liver representative dynamic data. The final processed dynamic curves were derived from the stack plots (left). The alanine and lactate levels were similar in the normal rat liver case (top right), but (more ...)
Fig. 5 Comparison of representative liver slice spectra from 3D-MRSI acquisitions on normal (left) and fasted (right) rats. The spectra from normal liver showed similar levels of 13C-lactate and 13C-alanine. The spectra from fasted liver showed relatively lower (more ...)