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Metabolism of hyperpolarized [1-13C]pyruvate in awake, isoflurane and urethane anesthetized rat brain1Biomedical Imaging Unit, Department of Neurobiology, A. I. Virtanen Institute, University of Eastern Finland, Kuopio, Finland, 2Technical University of Denmark, Kongens Lyngby, Denmark
Synopsis
Pre-clinical MRI/MRS routinely uses anesthesia, which alters hemodynamics and metabolism. Here, we used hyperpolarised [1-13C]pyruvate to compare brain metabolism under isoflurane or urethane anesthesia and in awake rats. Spectroscopic [1-13C]pyruvate time courses were measured in sequence in awake, isoflurane and urethane anesthetized rats. Bicarbonate- and lactate-to-total carbon ratios decreased in order from awake animals to urethane to isoflurane anesthetized animals. No change was observed in bicarbonate-to-lactate ratio between the groups, however. The study shows dDNP experiments can be performed in awake rats thus avoiding issues related to anesthesia. However, the ratios between intracellular metabolites did not alter in awake rats.
Introduction
Dissolution dynamic nuclear polarization (dDNP) MRSI could offer a more direct way to study brain metabolism, although blood-brain-barrier permeability poses a significant challenge to most commonly used metabolites1. Furthermore, anesthesia is used routinely in pre-clinical MRI/MRS to reduce the motion artifacts and stress felt by the animals but this also alters underlying hemodynamics and metabolism. For example, the most commonly used inhalant anesthetic, isoflurane, has dose-dependent vasodilation effect on brain vasculature, and widely varied responses can be seen in functional MRI and electrophysiology with different anesthetics2-5. This has led to efforts of performing pre-clinical rodent studies without anesthesia6. Here, we used hyperpolarised [1-13C ]pyruvate to compare rat brain metabolism under isoflurane anesthesia, urethane anesthesia and in awake rats.Methods
Brain metabolism of hyperpolarized pyruvate was monitored in awake, isoflurane (1.3-1.8 %), and urethane (1500 mg/kg) anesthetized rats. Nine female Wistar rats (weight 247-334 g) were habituated for awake MR experiments using a four-day habituation protocol described by Stenroos et al.7 with real MR performed on day 5. In awake experiments, shimming and slice selection were performed under isoflurane anesthesia after which the animal was allowed to wake up inside the magnet. 13C experiment was performed five to ten minutes after the animal had woken up. Only 13C spectroscopy was used because dynamic 13C spiral imaging sequence was deemed to be too loud for awake animals. 13C experiments were repeated under isoflurane anesthesia at day 8 and under terminal urethane anesthesia at day 10. Temperature and breath rates of animals were monitored during the experiments.
[1-13C]pyruvic acid was hyperpolarized with radical AH11501 at 1.35 K, 6.7 T and 188 GHz for 1.5 h in experimental HYPERMAG hyperpolarizer (DTU, Denmark)8. The sample was dissolved with 0.2 M Tris buffer, neutralized with 1 M NaOH and injected through tail vein (0.7 μmol/kg) to an animal inside the magnet. Slice-selective 13C spectra (TR = 1 s, nominal flip angle 10°, 10 mm axial slice centered 6 mm behind the olfactory bulb) were collected at 9.4 T using a 13C/1H transmit/receive surface coil (Neos Biotech, Spain). Peak integrals were estimated and metabolite ratios as well as fits to two-site exchange model were calculated.
Results
Average breath rates per minute were 149 ± 24, 61 ± 10 and 119 ± 20 in awake, isoflurane anesthetized and urethane anesthetized animals, respectively. Good quality 13C spectra with minimal movement artifacts were obtained in all groups confirming that awake protocol was suitable also for dDNP experiments involving tail injections (Figure 1). Bicarbonate-to-total carbon and lactate-to-total carbon ratios and corresponding exchange rates (kPL, kPC) decreased in order from awake animals to urethane to isoflurane anesthetized animals (Figure 2). In contrast, no change was observed in bicarbonate-to-lactate ratio between the groups.Discussion
The study shows that dDNP experiments can be performed in awake rats thus avoiding issues related to anesthesia. Marked differences in ratios between lactate and bicarbonate to total carbon signal, which reflects the inflow of pyruvate precursor, and in the corresponding exchange rates were observed between groups. While this effect may have some contribution from increased pyruvate uptake, it is likely mainly due to anesthesia-related alterations in CBF and CBV, e.g. extensive vasodilation under isoflurane anesthesia leading to relatively larger pyruvate pool, as reported previously1,9. Indeed, our Bic/Lac ratios are in good agreement with isoflurane anesthesia experiments by Josan et al.1. Our results also agree with Paasonen et al.10, who reported that urethane anesthesia resembles the awake state more than isoflurane anesthesia. Differences in anesthesia state were not reflected into ratios between intracellular metabolites, however. This suggests that either resting-state brain metabolism is not sufficiently altered between awake and anesthetized rats to be detected using the chosen approach or that some other factor, for example blood-brain-barrier permeability, confounds the results. Previous studies indicate that blood-brain-barrier permeability is the limiting factor in brain studies using hyperpolarized pyruvate1,9.Conclusion
Despite marked differences in hemodynamics between awake and anesthetized animals, no difference in metabolism, as assessed using a ratio of intracellular metabolites bicarbonate and lactate, was observed.Acknowledgements
This research was funded by Academy of Finland (project grant 286895). The hyperpolariser was funded by Academy of Finland (FIRI, Biocenter Finland infrastructure) and Business Finland/EU Regional Development Fund.References
1. Josan S, Hurd R, Billingsley K, et al. Effects of isoflurane anesthesia on hyperpolarized (13)C metabolic measurements in rat brain. Magn Reson Med. 2013;70(4):1117-1124.
2. Todd M, Weeks J. Comparative effects of propofol, pentobarbital, and isoflurane on cerebral blood flow and blood volume. J Neurosurg Anesthesiol. 1996 Oct;8(4):296-303.
3. Conzen P, Vollmar B, Habazetti H, et al. Systemic and regional hemodynamics of isoflurane and sevoflurane in rats. Anesth Analg. 1992 Jan;74(1):79-88.
4. Paasonen J, Salo R, Shatillo A, et al. Comparison of seven different anesthesia protocols for nicotine pharmacologic magnetic resonance imaging in rat. Eur Neuropsychopharmacol. 2016;26(3):518-531.
5. Marjańska M, Shestov A, Deelchand D, et al. Brain metabolism under different anesthetic conditions using hyperpolarized [1-13C]pyruvate and [2-13C]pyruvate. 2018;e4012
6. King J, Garelick T, Brevard M, et al. Procedure for minimizing stress for fMRI studies in conscious rats. J. Neurosci. Methods. 2005;148(2):154–160
7. Stenroos P, Paasonen J, Salo R, et al. Awake Rat Brain Functional Magnetic Resonance Imaging Using Standard Radio Frequency Coils and a 3D Printed Restraint Kit. Front Neurosci. 2018 Aug 20;12:548.
8. Ardenkjær-Larsen J, Bowen S, Petersen J, et al. Cryogen-free dissolution dynamic nuclear polarization polarizer operating at 3.35 T, 6.70 T, and 10.1 T. Magn Reson Med. 2018 Oct 25
9. Miller J, Grist J, Serres S, et al. 13C Pyruvate Transport Across the Blood-Brain Barrier in Preclinical Hyperpolarised MRI. Sci rep. 2018;8:15082
10. Paasonen J, Stenroos P, Salo R, et al. Functional connectivity under six anesthesia protocols and the awake condition in rat brain. NeuroImage. 2018;172:9-20.
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