Info
Background:
13C hyperpolarized imaging can generate metabolic contrast at clinical and pre-clinical levels. Given the imperative role of pre-clinical models to test new hypotheses prior to clinical translation, it is important to establish paradigms that adequately recapitulate conditions at the clinical level. One major limitation is the use of anesthetics in pre-clinical imaging. Anesthetics are known to drastically perturb circulation, respiration, and metabolism1, potentially limiting the ability to extrapolate findings to future clinical studies. Given the success of awake imaging protocols in fMRI1,2, similar strategies have been adapted for 13C hyperpolarized imaging. While these studies detected enhanced metabolism in awake animals, they were limited to lengthy acclimation protocols, surgical implantation of restraints, spectroscopy, and low-resolution images3,4.
Hypothesis/Objective:
Here, we hypothesized that these limitations could be resolved by utilizing a noninvasive 3D-printed restrainer, a streamlined acclimation protocol, and high-resolution dynamic imaging to assess data quality between acclimated and non-acclimated animals.
Methods:
First, we designed a 3D-printed restrainer that incorporates a spine immobilizer coupled to a cranial crown and a fitted cradle to allow for unperturbed respiration while maintaining head and body position. We then established an acclimation protocol by anesthetizing animals (2-3% isoflurane), applying the restrainer, maintaining them anesthetized for 5 minutes, turning off anesthesia, and maintaining them awake for 2.5 minutes in a dark enclosure prior to anesthetization and removal. Acclimation occurred 2x a day for 5 days before imaging. Non-acclimated animals remained in standard housing with periodic exposure to handlers. Motion was assessed with T2-weighted imaging before, during, and after awake periods. Once acclimated, animals were tail-vein catheterized, anesthetized, restrained, and placed in a 3T MRI scanner. After anatomical imaging, anesthesia was stopped, animals were allowed to waken (>120 bpm), followed by a 30s infusion of 200 mL of ~100 mM of hyperpolarized [1-13C]pyruvate generated with DNP. A dynamic 2D EPSI sequence was started at the same time as the injection with the following parameters: 10 mm axial slice, 32×32 matrix (1.25 mm2 resolution), 75 kHz bandwidth, 15° flip angle, 62.5 ms TE, and 2 s TR. Images were processed with an in-house MATLAB (R2024b) script that denoised, phased, line broadened, fitted peaks, and generated metabolite maps. Dynamic plots were based on down-sampled ROIs covering the brain to quantitate mean intensity on native resolution maps and regional ROIs on interpolated maps.
Results:
Non-acclimated animals showed distinct motion artifacts while awake and had significantly higher mean-voxel displacement of their brains compared to acclimated animals. Animal stress was then assessed by plasma corticosterone concentrations which showed all groups responded near or within baseline levels5. 13C metabolite maps demonstrate distinct signals highlighting specific brain regions, vasculature, and muscles in acclimated animals, whereas signal appeared more diffused in non-acclimated animals. Quantitatively, our results demonstrate considerably less variability in the brain scans of acclimated animals compared to non-acclimated animals. Similarly, regional analysis of the cortex and central brain region covering forebrain, midbrain, hindbrain, and cerebellum (FMHC) across the slice acquired, showed lower standard deviations and significantly greater lactate intensity in the FMHC of acclimated animals, which is likely attributed to higher MCT1 expression in the cerebellum compared to the cortex6. Lastly, reduced motion of acclimated animals allowed for the detection of a 6M urea phantom during the same hyperpolarized imaging scan, which permitted the reconstruction of concentration-based images and kinetic maps of kPL using published models7.
Conclusion:
Overall, these data demonstrate that awake animal imaging with noninvasive restraints may be useful for achieving higher quality 13C MRI data that can be utilized to generate quantitative concentration estimates of metabolic activity.
Author
Mario C. Chang
Memorial Sloan Kettering Cancer Center