Activated mononuclear phagocytes (MPs, macrophages/microglia) are key players in the progression of multiple sclerosis (MS). Interestingly, upon activation, MPs undergo metabolic reprogramming, and especially increase their lactate production^1,2. Importantly, a recent in vitro study shows that increased lactate production resulted from inhibitory phosphorylation of the pyruvate dehydrogenase (PDH) complex by the pyruvate dehydrogenase kinase 1 (PDK1) enzyme³. Based on this new knowledge, we questioned if hyperpolarized ¹³C Magnetic Resonance Spectroscopic Imaging (HP ¹³C MRSI) of pyruvate⁴ could be used to assess increased pyruvate to lactate conversion linked to MPs activation in MS (Fig.1A). To do so, we longitudinally studied the toxin-induced cuprizone (CPZ) mouse model, a well established MS model⁵. We demonstrated that HP ¹³C MRSI of pyruvate could detect MPs activation non-invasively during disease progression, thus highlighting its potential for monitoring neuroinflammation levels in MS. We further demonstrated, for the first time in vivo, that the detected increase HP lactate production was linked to PDK1-induced PDH inhibition in activated MPs.

Animals: C57BL/6J mice (n=5) received a 0.2% CPZ diet for 6 weeks while aged-matched control (CTRL) received a standard rodent chow (n=8) ⁶. All CTRL and CPZ fed mice were imaged prior (W0) and after 4 and 6 weeks of CPZ (W4 CPZ, W6 CPZ) (Fig.1B).

MR acquisitions: T2-weighted images were acquired for proper assessment of the corpus callosum (CC) location, region of maximum inflammation in the CPZ model (TE/TR=20/1200ms, thickness=0.5mm, NA=2, matrix=256x256, FOV=30x30mm²). For ¹³C MRS, 24μL of [1-¹³C] pyruvate preparation was hyperpolarized using a Hypersense DNP polarizer (Oxford Instruments) for one hour³. After dissolution, HP pyruvate was rapidly dissolved in isotonic buffer (pH~7) and injected intravenously (iv) over 12sec. From the beginning of iv injection, 2D dynamic CSI ¹³C data were acquired on a 14.1T MR system using: TE/TR=1.2/60ms; SW 2500Hz; 128points; 4sec resolution; FA=10deg; FOV=24x24 mm²; 5mm thickness.

Immunofluorescence (IF): IF analyses were performed for myelin (MBP), MPs (Iba-1) and pyruvate dehydrogenase kinase-1 (PDK1), (n=3 mice per group). Pyruvate dehydrogenase (PDH) activity in the CC was assessed using a PDH activity assay kit (Abcam), (n=4 mice per group).

Data analysis: HP lactate and HP pyruvate levels were calculated as the sum of integrals over time (Fig.1C). For both MR and IF data, CPZ and CTRL groups were compared using unpaired t-tests and longitudinal analyses of HP ¹³C MRSI using paired t-tests (*p<0.05, **p<0.01, ***p<0.001).

T2-weighted images showed hyperintensity in the CC region in W4 and W6 CPZ animals (arrows), confirming the expected effect of the diet (Fig.2A). IF analysis demonstrated that hyperintensity was linked to modulations in myelin (MBP) and MPs activation (Iba-1) levels (Fig.2B). Importantly, even though W4 and W6 lesions are linked to different underlying cellular events, (W4: high demyelination/high inflammation; W6: partial remyelination/low inflammation), T2-weighted could not distinguish between W4 and W6 animals, highlighting the fact that this method is not specific to inflammation.

In contrast, HP ¹³C MRS of pyruvate showed specificity to neuroinflammatory events only. While the pyruvate delivery remained stable over time in all experimental groups (Fig.3A), the HP lactate/pyruvate ratio was significantly increased at W4 in the CC of CPZ animals as compared to age-matched CTRL (72±29% at W4 CPZ, p=0.019, n=5) (Fig.3B, C, D and E). This ratio was then significantly decreased by 20±14% at W6 CPZ (p=0.028, n=5, Fig.3F).

Importantly, IF analyses confirmed that increased HP lactate/pyruvate ratio was associated with a strong upregulation of PDK1 in activated MPs at W4 CPZ, as demonstrated by the merged image (p<0.0001, n=3), (Fig.4A-B). Importantly, subsequent inhibition of PDH activity was also measured in the CC (p<0.0001, n=4), (Fig.4C). Further in line with the HP results, at W6 CPZ, the number of PDK1-expressing MPs present in the CC significantly decreased (p=0.001, n=3) and PDH activity subsequently increased as compared to 4W CPZ.

All together, our results demonstrate that ¹³C MRSI of HP pyruvate can detect increased lactate production in vivo in a preclinical MS model, and that this increase is linked to the presence of activated MPs that upregulate PDK1 and subsequently inhibit PDH activity, thus pushing the flux towards lactate production. Importantly, this report is the first to demonstrate the use of a metabolic imaging method to monitor neuroinflammation. Because HP ¹³C MRSI is clinically translatable and expanding rapidly, this study is of high significance for future clinical trials not only on MS, but also all neurological diseases presenting an inflammatory component. Such method would enhance diagnosis and help refine therapeutic regimen, which will ultimately improve clinical outcome and patient care.