Stroke is the third leading cause of death worldwide and the leading cause of disability in the adult. It is therefore a major public health challenge in the context of the current demographic changes. Among a wide range of applications, hyperpolarized (HP) magnetic resonance enables in vivo real-time measurement of biochemical transformations of hyperpolarized ¹³C-labeled precursors ¹, including lactate, a known neuroprotectant in stroke at the preclinical level ^2,3. The aim of this study was therefore to demonstrate the feasibility of probing lactate metabolism in vivo using hyperpolarized ¹³C-lactate, to understand its biodistribution and its effect on metabolism and neuroprotection in an animal stroke model.A frozen mixture of sodium L- [1-¹³C]lactate solution (45-55% (w/w) in H2O, 99% ¹³C) and d8-glycerol (1:1 w/w) doped with 50mM TEMPOL radical was hyperpolarized for 2 hours in a 7 T custom-designed polarizer (196 GHz / 1.00±0.05 K)⁴. In parallel, 30 min focal ischemia was induced in C57BL/6 male mice by inserting a silicon coated suture through the common carotid artery (CCA) into the internal carotid artery (ICA) and advancing it into the arterial circle to occlude the origin the middle cerebral artery (MCA) under laser Doppler cerebral blood flow (CBF) monitoring². The CBF was restored by withdrawing the filament. Animals were placed into a 9.4 T/ 31 cm actively shielded animal scanner (Varian/Magnex) equipped with a home-built quadrature ¹H - single loop ¹³C surface coil. Adjustment and Shimming procedure (FASTMAP) were performed prior to T₂-weighted MRI (Figure 1) and ¹H MRS in a voxel localized in the ischemic area (Figure 2). Metabolites were then quantified using LC Model. 350 μL of hyperpolarized [1-¹³C]lactate solution were finally injected within 5 s through the femoral vein. The ¹³C MR spectrum was then acquired every 3 s starting at the time of injection. Lactate concentration in the plasma was quantified from venous blood just after reperfusion and after hyperpolarized lactate injection.T_2w images presented in Figure 1 depict typical edema clearly visible in the striatum about 2h after ischemia. Endogenous lactate quantification from ¹H MRS shows dramatic increase in the ischemic region compared to sham (Figure 3A), in good agreement with previous studies⁵. Infusion of hyperpolarized [1-¹³C] lactate lead to ¹³C labeling of the pyruvate pool. It has been demonstrated that the kinetic rates obtained from fitting the evolution of the signals of HP [1-¹³C]pyruvate and [1-¹³C]lactate are directly proportional to ratios between the summed signals ⁶. The pyruvate-to-lactate ratio was then calculated from the peak integrals of the summed spectra. In the MCAO mice the labeling of the pyruvate pool was about two times higher compared to sham (Figure 3B).It has been demonstrated that the ¹³C pyruvate signal measured following the injection of hyperpolarized ¹³C-lactate is directly related to the endogenous pyruvate pool size, the pyruvate pool size being labeled by exchange⁷. The detected pyruvate-to-lactate ¹³C signal ratio is representative of the local pyruvate-to-lactate concentration ratio. Despite the intrinsic limited sensitivity of measuring globally ¹³C MR signals in the whole brain, it clearly shows differences between ischemic and non-ischemic brains at this early stage after ischemic onset. Localized acquisitions will definitely improve the accuracy and relevance of these first preliminary results. L-lactate offers neuroprotection in ischemia most likely by acting as both an HCA1 receptor agonist for non-astrocytic cells as well as an energy substrate³. Performing repeated HP lactate injections and ¹³C MR measurements at different time points after the ischemia onset could help to understand its in vivo biodistribution and its effect on metabolism in stroke, while exerting neuroprotection.This preliminary study shows that it is feasible to measure lactate metabolism in vivo in a mouse model of stroke following intravenous injection of hyperpolarized L-[1-¹³C]lactate, suggesting new perspectives to understand its in vivo biodistribution and its neuroprotective effect in an animal stroke model.