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Kinetic Modeling of Hyperpolarized [1-13C]Lactate Metabolism in a Mouse Model of Ischemic Stroke
Thanh Phong Lê1,2, Lara Buscemi3, Elise Vinckenbosch1, Mario Lepore4, Rolf Gruetter 2,5,6, Lorenz Hirt 3, Jean-Noël Hyacinthe 1,7, and Mor Mishkovsky 2

1Geneva School of Health Sciences, University of Applied Sciences and Arts Western Switzerland (HES-SO), Geneva, Switzerland, 2Laboratory of Functional and Metabolic Imaging, École polytechnique fédérale de Lausanne (EPFL), Lausanne, Switzerland, 3Department of Clinical Neurosciences, Centre hospitalier universitaire Vaudois (CHUV), Lausanne, Switzerland, 4Centre d'Imagerie Biomédicale (CIBM), École polytechnique fédérale de Lausanne (EPFL), Lausanne, Switzerland, 5Department of Radiology, University of Geneva (UNIGE), Geneva, Switzerland, 6Department of Radiology, University of Lausanne (UNIL), Lausanne, Switzerland, 7Image Guided Intervention Laboratory, University of Geneva (UNIGE), Geneva, Switzerland

Synopsis

Stroke is the second cause of death and third leading cause of disability worldwide. Lactate injection was found to provide neuroprotection in preclinical models of ischemic stroke.

Alteration of the metabolism induced by ischemia can be measured in real time using magnetic resonance with hyperpolarized 13C labeled probes.

This study aims at investigating the feasibility of quantifying changes in the kinetics of hyperpolarized [1-13C]lactate metabolism following ischemia in a mouse model of stroke in order to assess the potential of hyperpolarized lactate as a theranostic agent for stroke.

Introduction

Stroke is the second cause of death, and the third leading cause of disability worldwide.1 Ischemic stroke represents 80% of strokes and can be treated by restoring the obstructed blood flow by either thrombolysis or thrombectomy within respectively 4.5h and 7.3h after onset of ischemia.2,3

Neuroprotective strategies could extend this narrow time window and improve patient rehabilitation. In preclinical studies, lactate administered after reperfusion from ischemic stroke provides neuroprotection, reduces brain cell death and improves the neurological outcome.4,5

Hyperpolarized (HP) [1-13C]lactate prepared with dynamic nuclear polarization6 (DNP) can be employed for studying real time in vivo metabolism.7,8,9 It has been shown that lactate metabolism was modulated in a transient mouse model of stroke, modifying the labeling of the [1-13C]pyruvate pool from HP [1-13C]lactate in function of the time elapsed after reperfusion.10

This study aims at demonstrating the feasibility of quantifying cerebral lactate metabolism kinetics following infusion of HP [1-13C]lactate in a mouse model of stroke. Combining these potential diagnostic findings with the neuroprotective effect of lactate could pave the way to a theranostic approach for stroke.

Methods

Hyperpolarization: A frozen mixture of sodium L-[1-13C]lactate, H2O, glycerol and OX63 radical was hyperpolarized in a custom-designed 7T/1K DNP polarizer11, resulting in a liquid-state polarization of (35.7±11.5)%.

Mouse middle cerebral artery occlusion (MCAO) model of stroke: C57BL6/J male mice (6-10 weeks) were anesthetized using 1.5-2% isoflurane in air/O2 (1:1). A focal ischemic lesion in the left striatum was induced by occluding the middle cerebral artery with a silicon-coated filament. The filament was withdrawn after 30min to allow reperfusion. The regional cerebral blood flow (rCBF) was monitored throughout the surgery by Laser-Doppler flowmetry. Animals were included in the study only if the rCBF dropped by 80% during occlusion and raised above 50% of baseline within 10min after reperfusion. A femoral vein was cannulated during occlusion to posteriorly inject the lactate. Sham operated mice underwent the same procedure without any artery ligation or suture insertion.

Acquisition: Upon reperfusion, mice were placed into a 9.4T/31cm horizontal bore MRI scanner (Varian/Magnex) with a home-built 1H quadrature/13C single loop coil above the head. At 1h post-reperfusion in MCAO mice (n=2) and 1h post-surgery in sham-operated animals (n=2), 325μL of ≈90mM HP [1-13C]lactate solution was injected and 13C MR spectrum was acquired every 3s with 30° BIR-4 adiabatic pulses.

Kinetic modeling: Spectra were fitted with jMRUI12,13,14 (v5.2.1) to obtain the time course of metabolites signal. From a simplified scheme of [1-13C]lactate cerebral metabolism (Fig.1a), a kinetic model (Fig.1b) was derived as follows:

  • Steps were modeled as first-order reactions.
  • Different rate constants were assigned to opposite directions of reversible reactions.
  • Since lactate dehydrogenase (LDH) activity is fast compared to lactate transport across the blood brain barrier9, both were simplified into a single step.
  • Conversion of pyruvate up to bicarbonate was accounted in a single step since 13CO2 was not detected due to limited bandwidth of radio-frequency pulses.

Kinetic rate constants were determined from fitting the kinetic model on individual time courses using Levenberg-Marquart algorithm. Monte-Carlo simulations tested precision and accuracy of the model.


Results

In anatomical T2W images (Fig.2), the striatal ischemic lesion slightly apparent at 1h post-reperfusion becomes clearly visible at 2h post-reperfusion. Following infusion, [1-13C]lactate is metabolized into [1-13C]pyruvate, [1-13C]alanine and [13C]bicarbonate (Figs.3-4).

Kinetic modeling suggests lower lactate to pyruvate (kLP) and higher pyruvate to bicarbonate (kPB) rate constants in MCAO 1h post-reperfusion compared to sham (Fig.5a). The pyruvate/lactate ratio (PLR) and bicarbonate/pyruvate ratio (BPR) tend to respectively decrease and increase after MCAO (Fig.5b). In these preliminary results, high variability prevents observation of trends in kPA, backwards and elimination rate constants (Fig.5c).

Discussion

MCAO and sham at 1h post-reperfusion/surgery show different dynamic trends of lactate to pyruvate and pyruvate to bicarbonate conversion while T2W images do not show clear morphological changes.

Lower kLP might be related to the increased endogenous lactate concentration after MCAO.15 Higher kPB implies increased mitochondrial activity resulting from greater energy demand after stroke. Trends of kLP and kPB are consistent with decreased PLR and increased BPR in MCAO from a model-free approach to kinetic analysis.16

To improve the accuracy of the kinetic model in the MCAO case, physiological changes such as the increase of monocarboxylate transporters expression17 and the increase of endogenous lactate concentration15 should be explicitly considered.

Although the limitations of our measurement, distinct metabolic kinetics of HP [1-13C]lactate between MCAO and sham demonstrate its potential as a MR molecular imaging contrast for stroke.

Conclusion

The metabolic kinetics of the HP neuroprotective agent [1-13C]lactate was quantified in a mouse model of stroke, and shows different dynamics compared to sham.

Acknowledgements

The authors gratefully thank Dr. Analina Da Silva and Dr. Stefan Mitrea for their assistance in the animal preparation. This study is supported by the Swiss National Science Foundation (310030_170155), the Centre d’Imagerie Biomédicale of the University of Lausanne, École polytechnique fédérale de Lausanne, University of Geneva, Geneva University Hospitals, Lausanne University Hospital, and the Leenaards and Louis-Jeantet Foundations.

References

1. Johnson W, Onuma O, Owolabi M, Sonal S, Stroke: a global response is needed. Bulletin of the World Health Organization. 2016;94:634-634A

2. The IST-3 collaborative group. The benefits and harms of intravenous thrombolysis with recombinant tissue plasminogen activator within 6 h of acute ischaemic stroke (the third international stroke trial [IST-3]): a randomised controlled trial. The Lancet. 2012;379(9834):2352-2363

3. Saver JL, Goyal M, van der Lugt A, et al. Time to Treatment With Endovascular Thrombectomy and Outcomes From Ischemic Stroke: A Meta-analysis. JAMA. 2016;316(12):1279-1289.

4. Castillo X, Rosafio K, Wyss MT, et al. A probable dual mode of action for both L- and D-lactate neuroprotection in cerebral ischemia. J Cereb Blood Flow Metab. 2015;35(10):1561-1569.

5. Berthet C, Lei H, Thevenet J, et al. Neuroprotective Role of Lactate after Cerebral Ischemia. J Cereb Blood Flow Metab. 2009;29(11):1780–1789

6. Ardenkjær-Larsen JH, Fridlund B, Gram A, et al. Increase in signal-to-noise ratio of > 10,000 times in liquid-state NMR. Proc. Natl. Acad. Sci. 2003;100(18)10158-10163

7. Chen AP, Kurhanewicz J, Bok R, et al. Feasibility of using hyperpolarized [1-13C]lactate as a substrate for in vivo metabolic 13C MRSI studies. Magn Reson Imaging. 2008;26(6):721-726.

8. Bastiaansen JAM, Yoshihara HAI, Takado Y, et al. Hyperpolarized 13C lactate as a substrate for in vivo metabolic studies in skeletal muscle. Metabolomics. 2014;10(5):986-994

9. Takado Y, Cheng T, Bastiaansen JAM, et al. Hyperpolarized 13C Magnetic Resonance Spectroscopy Reveals the Rate-Limiting Role of the Blood–Brain Barrier in the Cerebral Uptake and Metabolism of L-Lactate in Vivo. ACS Chem. Neurosci. 2018. 10.1021/acschemneuro.8b00066

10. Hyacinthe JN, Buscemi L, Lepore M, et al. Evaluating hyperpolarized lactate as a theranostic agent for stroke. Proc. Intl. Soc. Mag. Reson. Med. 26. 2018:3708

11. Cheng T, Capozzi A, Takado Y, et al. Over 35% liquid-state 13C polarization obtained via dissolution dynamic nuclear polarization at 7 T and 1 K using ubiquitous nitroxyl radicals. Phys. Chem. Chem. Phys. 2013;15:20819-20822

12. Naressi A, Couturier C, Devos J, et al. Java-based graphical user interface for the MRUI quantitation package. Magma: Magnetic Resonance Materials in Physics, Biology, and Medicine. 2001;12(2-3):141–152

13. Stefan D, Cesare FD, Andrasescu A, et al. Quantitation of magnetic resonance spectroscopy signals: the jMRUI software package. Measurement Science and Technology. 2009;20(10):104035.

14. Vanhamme L, Van den Boogaart A, Van Huffel S. Improved method for accurate and efficient quantification of MRS data with use of prior knowledge. Journal of Magnetic Resonance. 1997;129:35-43

15. Lei H, Berthet C, Hirt L, Gruetter R. Evolution of the Neurochemical Profile after Transient Focal Cerebral Ischemia in the Mouse Brain. J Cereb Blood Flow Metab. 2009;29(4):811-819

16. Hill DK, Orton MR, Mariotti E, et al. Model Free Approach to Kinetic Analysis of Real-Time Hyperpolarized 13C Magnetic Resonance Spectroscopy Data. PLOS ONE. 2013;8(9):e71996

17. Rosafio K, Castillo X, Hirt L, et al. Cell-specific modulation of monocarboxylate transporter expression contributes to the metabolic reprograming taking place following cerebral ischemia. Neuroscience. 2016;317:108-120

Figures

Fig. 1. (a) Simplified schematics of [1-13C]lactate metabolism. Lactate crosses the blood-brain barrier (BBB) via monocarboxylate transporters (MCTs). Intracellular [1-13C]lactate exchanges to [1-13C]pyruvate via lactate dehydrogenase (LDH). [1-13C]pyruvate is either converted into [1-13C]alanine by alanine aminotransferase (ALT) or transported into the mitochondria via mitochondrial pyruvate carriers (MPCs), then oxidized by pyruvate dehydrogenase (PDH), producing 13CO2 remaining in equilibrium with [13C]bicarbonate via carbonic anhydrase (CAT). (b) Kinetic model of cerebral [1-13C]lactate metabolism. [1-13C]lactate signal is used as the input function. Lac*, Ala*, Pyr* and Bic* denote the time-varying signal amplitude of the corresponding 13C metabolites.

Fig. 2. T2W axial images of the brain acquired in sham (a, c) and in MCAO (b, d) at 1h or 2h post-surgery or post-reperfusion. In the MCAO mouse, the lesion is slightly visible at 1h after reperfusion (b) and becomes clearly apparent in the image acquired at 2h post-reperfusion (d). The sham animal does not present any lesion as expected (a, c). Images were acquired with a FSEMS sequence (voxel size: 0.07x0.07x1mm3, TR = 4s, effective TE: 52ms, 2 dummy scans, 4 averages).

Fig. 3. Typical cerebral 13C MRS dynamic spectra measured after infusion of HP [1-13C]lactate (183.5ppm) in a mouse 1h after sham surgery (a) and a mouse with MCAO surgery 1h post-reperfusion (b). Spectra were normalized to the maximal lactate signal. [1-13C]pyruvate (171.3ppm), [1-13C]alanine (176.9ppm) and [13C]bicarbonate (161.4ppm) are observed in both cases. The red spectrum is the summed spectrum of the first 60s post-infusion. Peaks at 177.7ppm (*) and 179.5ppm (**) are impurities in the lactate solution. T2W images in inset were acquired ≈10min before injection.

Fig. 4. Representative in vivo time course of the cerebral MR signal from Fig.3. Dots represent the experimental data while solid lines are the fitted metabolic time courses from the kinetic model. The signal amplitude was normalized to the maximal lactate signal.

Fig. 5. (a) Averaged kinetic rate constants from metabolic modelling in 2 MCAO and 2 sham experiments. Error bars denote the largest value of either the group’s standard deviation, or half the square root of the sum of the variance of individual experiments. Trends of lower lactate to pyruvate (kLP) and higher pyruvate to bicarbonate (kPB) kinetic rate constants are observed in MCAO compared to sham. (b) Ratios of areas under the curve (AUC) in the first 60s after injection. Pyruvate/lactate and bicarbonate/pyruvate AUC ratios seem respectively lower and higher in MCAO compared to sham. (c) Backwards and elimination rate constants.

Proc. Intl. Soc. Mag. Reson. Med. 27 (2019)
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