3708

Evaluating hyperpolarized lactate as a theranostic agent for stroke

Jean-Noël Hyacinthe^1,2, Lara Buscemi³, Mario Lepore⁴, Rolf Gruetter^4,5,6,7, Lorenz Hirt³, and Mor Mishkovsky⁵

¹School of Health Sciences - Geneva, University of Applied Sciences and Arts Western Switzerland, Geneva, Switzerland, ²Image Guided Intervention Laboratory, Faculty of Medicine, University of Geneva, Geneva, Switzerland, ³Department of Clinical Neurosciences, Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland, ⁴Centre d'Imagerie Biomédicale (CIBM), Ecole Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland, ⁵Laboratory of Functional and Metabolic Imaging, Ecole Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland, ⁶Department of Radiology, University of Geneva, Geneva, Switzerland, ⁷Department of Radiology, University of Lausanne, Lausanne, Switzerland

Synopsis

Stroke is the leading cause of disability and the third leading cause of death worldwide. Lactate administration into the ischemic brain directly after blood reperfusion was found to be neuroprotective. MR with hyperpolarized (HP) probes enables in vivo real-time measurement of biochemical transformations of HP ¹³C-labeled precursors, including lactate. The aim of this study was to demonstrate the feasibility of probing HP [1-¹³C]L-lactate metabolism in mice brain after ischemic stroke, and to study the influence of the time window after reperfusion on its conversion in order to evaluate the potential of HP lactate as a theranostic agent for stroke.

INTRODUCTION

Stroke is the leading cause of disability and the third leading cause of death worldwide. 80% of strokes are of the ischemic subtype, which can be treated within a narrow time window of 4.5-6 hr from the ischemic onset by restoring blood flow to the ischemic brain^1,2. Consequently, only limited number of stroke patients can receive treatment. Despite promising preclinical results of neuroprotective strategies, there are currently no successful bench-to-bedside translations. It has been shown that lactate administration directly after blood reperfusion protects against ischemia-induced cell death and disability^3,4. In a clinical pilot study L-lactate was safely administered to traumatic brain injury patients⁵. Hyperpolarization techniques boost MR sensitivity and enable in vivo real-time measurement of biochemical transformations of hyperpolarized (HP) ¹³C-labeled precursors, including lactate⁶. The aim of this study was to demonstrate the feasibility of measuring HP [1-¹³C]L-lactate metabolism in mouse brain after ischemic stroke, and to study its conversion to pyruvate at different times after reperfusion in order to evaluate the potential of HP lactate as a theranostic agent for stroke.

METHODS

Frozen beads containing sodium [1-¹³C]L-lactate solution (45-55% (w/w) in H₂O) and d₈-glycerol (1:1 w:w) with 58 mM TEMPOL radical was dynamically polarized using a custom-designed 7T DNP polarizer (196 GHz/ 1.00±0.05K). C57BL/6J male mice (8 weeks, 25.2 ± 2.4 g) were anesthetized using isoflurane. A 30 min transient middle cerebral artery occlusion (MCAO) was induced as illustrated in Figure 1. Animals were included in the study if the regional cerebral blood flow (rCBF) was reduced to <20% of rCBF_initial during occlusion and increased to >50% of the initial rCBF_initial within 10 min after reperfusion onset. A cannula was inserted into the femoral vein for HP lactate infusion. In order to minimize the exposure to anesthesia, cannulation was performed during the ischemia. Upon reperfusion, animals were placed into a 9.4T/31cm actively shielded animal scanner (Varian/Magnex). MR measurements were performed using a home-built ¹H-quadrature/¹³C-single loop surface coil that was placed on top of the mouse head. B₀ inhomogeneity was corrected using FASTMAP protocol. Anatomical T_2W images were acquired before infusion of the HP solutions. 325 μL of 90 mM HP [1-¹³C]L-lactate solution was injected 1 hr or 2 hr post reperfusion (p.r.) in MCAO mice and 1hr post-surgery in sham operated animals. A series of pulse-acquire (30°, BIR-4 pulse) spectra were measured every 3s.

RESULTS

Anatomical T_2W images presented in Fig 2 illustrate minor morphological modifications at 1 hr p.r. The characteristic striatal lesion following MCAO is clearly visible at 2 hr p.r. As expected, images of sham animals show no lesion. Infusion of HP [1- ¹³C]L-lactate lead to the labeling of the pyruvate pool (Fig 3). The time evolution of this conversion was quantified, and demonstrated larger [1-¹³C]pyruvate signals 1 hr p.r. compared to 2 hr p.r. and sham (Fig 4). Pyruvate-to-lactate ratio (PLR) was then calculated from the summed spectra during the first 37.5 s of the experiment and was corrected to [1-¹³C]L-lactate concentration in blood at the time of injection for each individual animal (Fig 5). A significantly higher PLR was found 1 hr p.r. compared to 2 hr p.r. and sham (p < 0.05).

DISCUSSION

We successfully demonstrated the feasibility to measure the metabolism of a neuro-protective agent, i.e. [1-¹³C]L-lactate, in mice after ischemic stroke. A significantly higher PLR was found 1 hr p.r. compared to 2 hr p.r. and sham animals, implying a dependency of this ratio on the time elapsed from reperfusion. HP [1-¹³C]L-lactate crosses the BBB via monocarboxylate transporters (MCTs) and is converted to [1-¹³C]pyruvate through LDH-mediated exchange. Therefore the labelling of [1-¹³C]pyruvate is directly related to the endogenous pyruvate pool size⁷. Thus, the large PLR observed in our study may indicates larger endogenous pyruvate pool. Nonetheless, It has been reported that the BBB plays a limiting role in the observation of cerebral metabolism of HP monocarboxylic-acids^8,9. Given that MCT expression and distribution increase after MCAO in mice¹⁰, this should result in a larger concentration of HP [1-¹³C]L-lactate in the brain compartment that rapidly exchange to [1-¹³C]pyruvate. Further investigations are necessary to clarify the mechanism of this effect in our system. Regardless of the mechanism, it is important to note that higher PLR was measured when no clear morphological changes were depicted in the T_2W images, demonstrating the potential of HP [1-¹³C]lactate as a theranostic contrast agent for molecular imaging after stroke.

CONCLUSION

HP [1-¹³C]L-lactate metabolism can be measured in MCAO mouse stroke model and it is dependent on the time elapsed from reperfusion.

Acknowledgements

This work was supported by the Swiss National Science Foundation (grant #310030_170155), the Centre d’Imagerie BioMédicale (CIBM) of the UNIL, UNIGE, HUG, CHUV, EPFL, and the Leenards, Jeantet, Biaggi and Juchum Foundations.

References

1. Hacke, W. et al. Thrombolysis with alteplase 3 to 4.5 hours after acute ischemic stroke. The New England journal of medicine 359, 1317-1329, doi:10.1056/NEJMoa0804656 (2008).

2. Campbell, B. C. et al. Endovascular therapy for ischemic stroke with perfusion-imaging selection. The New England journal of medicine 372, 1009-1018, doi:10.1056/NEJMoa1414792 (2015).

3. Berthet, C. et al. Neuroprotective role of lactate after cerebral ischemia. Journal of cerebral blood flow and metabolism : official journal of the International Society of Cerebral Blood Flow and Metabolism 29, 1780-1789, doi:10.1038/jcbfm.2009.97 (2009).

4. Castillo, X. et al. A probable dual mode of action for both L- and D-lactate neuroprotection in cerebral ischemia. Journal of cerebral blood flow and metabolism : official journal of the International Society of Cerebral Blood Flow and Metabolism 35, 1561-1569, doi:10.1038/jcbfm.2015.115 (2015).

5. Ichai, C. et al. Sodium lactate versus mannitol in the treatment of intracranial hypertensive episodes in severe traumatic brain-injured patients. Intensive care medicine 35, 471-479, doi:10.1007/s00134-008-1283-5 (2009).

6. Chen, A. P. et al. Feasibility of using hyperpolarized [1-13C]lactate as a substrate for in vivo metabolic 13C MRSI studies. Magnetic resonance imaging 26, 721-726, doi:10.1016/j.mri.2008.01.002 (2008).

7. Kennedy, B. W., Kettunen, M. I., Hu, D. E. & Brindle, K. M. Probing lactate dehydrogenase activity in tumors by measuring hydrogen/deuterium exchange in hyperpolarized l-[1-(13)C,U-(2)H]lactate. Journal of the American Chemical Society 134, 4969-4977, doi:10.1021/ja300222e (2012).

8. Hurd, R. E. et al. Metabolic imaging in the anesthetized rat brain using hyperpolarized [1-13C] pyruvate and [1-13C] ethyl pyruvate. Magnetic resonance in medicine 63, 1137-1143, doi:10.1002/mrm.22364 (2010).

9. Hurd, R. E. et al. Cerebral dynamics and metabolism of hyperpolarized [1-(13)C]pyruvate using time-resolved MR spectroscopic imaging. Journal of cerebral blood flow and metabolism : official journal of the International Society of Cerebral Blood Flow and Metabolism 30, 1734-1741, doi:10.1038/jcbfm.2010.93 (2010).

10. Rosafio, K., Castillo, X., Hirt, L. & Pellerin, L. Cell-specific modulation of monocarboxylate transporter expression contributes to the metabolic reprograming taking place following cerebral ischemia. Neuroscience 317, 108-120, doi:10.1016/j.neuroscience.2015.12.052 (2016).

Figures

Middle cerebral artery occlusion (MCAO) was induced by inserting a silicon coated suture (green line) through the common carotid artery (CCA) into the internal carotid artery (ICA) and advancing it into the arterial circle to occlude the middle cerebral artery (MCA) resulting in a focal lesion in the striatum. The procedure was performed under laser Doppler-mediated cerebral blood flow (CBF) monitoring. CBF was restored by withdrawing the filament.

T_2W axial images of the brain of MCAO mice at different times after ischemia onset (A and B), illustrating the development of edema in the ischemic region compared to sham operated mice (C). Images acquired on different mice and ca. 5 min before the infusion of HP [1-¹³C]L-lactate with the following experimental parameters: FOV 18 x 18 mm with 1 mm slice thx, TR = 4 s, TEeffective = 52 ms, ds = 2, ns = 2.

Dynamic ¹³C MRS spectra measured in MCAO mouse head following the injection of HP [1-¹³C]L-lactate (183.5 ppm). [1-¹³C]pyruvate at 171.5 ppm can readily detected. In sum of the spectra shown on top panel the peak of ¹³C bicarbonate become visible at 161.5 ppm. Peak designated by (*) at 176 ppm results from chemical impurities that overlap with the expected [1-¹³C]alanine peak.

Representative in vivo time courses of [1-¹³C]L-lactate (black) and its metabolic product [1-¹³C]pyruvate (grey) in the mouse brain at different times after ischemia onset (A, B) and in sham operated mouse (C). Each time course was normalized to its maximal lactate signal respectively. The red dotted line was added to highlight the differences in [1-¹³C]pyruvate labelling.

Pyruvate-to-lactate (PLR) corrected to [1-¹³C]L-lactate concentration in blood at the time of injection (mean ± SEM) with individual data points. One-way ANOVA test indicates significant statistical difference (p < 0.05) between PLR 1 hr p.r. (black) to 2 hr p.r. (grey) and sham (red).

Proc. Intl. Soc. Mag. Reson. Med. 26 (2018)

3708