Traumatic brain injury (TBI) induces chronic activation of mononuclear phagocytes (microglia/macrophages, MPs), which persists for many years following initial insult. Interestingly, recently published data demonstrate a strong pro-inflammatory MPs response at acute and sub-acute time points after injury¹. Studies also show that modifying initial MPs response can rescue long term cognitive deficits, highlighting the critical and early role of pro-inflammatory MPs in TBI progression¹. In this context, non-invasive assessment of pro-inflammatory MPs status in vivo would be important for the development and validation of treatment strategies targeting TBI-dependent cognitive deficits.

Interestingly, MPs have been recently shown to undergo metabolic reprogramming upon activation, and especially increase their lactate production². In this study, we took advantage of this recently uncovered knowledge, and questioned the potential of hyperpolarized ¹³C Magnetic Resonance Spectroscopic Imaging (HP 13C MRSI³) of pyruvate to monitor MPs status in vivo. Our study demonstrates that HP lactate/pyruvate ratios as detected by ¹³C MRSI were significantly increased in TBI lesions as compared to normal brain at acute (12 and 24 hours) and sub-acute (7days) time points post cortical impact. This increased lactate/pyruvate ratio was associated with the presence of pro-inflammatory MPs, thus highlighting the potential of HP ¹³C MRSI to monitor neuroinflammation levels in TBI.

Animals: C57BL/6J mice (n=6) received TBI using the controlled cortical impact (CCI) model¹. Mice received a craniectomy ~3.5 mm in diameter at the coordinates: anteroposterior, -2.0 mm; mediolateral, +2.0 mm, with respect to bregma. The parameters for impact were for a contusion depth of 0.95 mm (from dura), velocity was constant at 4.0 m/s, and the impact was sustained for of 300 ms. Mice were imaged prior (Baseline) and after 12h, 24h, 7 days and 28 days post CCI (Fig.1A).

MR acquisitions: T2-weighted images were acquired for proper assessment of the TBI location (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 MPs (Iba-1), pyruvate dehydrogenase kinase-1 (PDK1) and nuclei (Hoechst), 48h post CCI.

Data analysis: HP lactate and HP pyruvate levels were calculated as the sum of integrals over time (Fig.1B). HP lactate/ pyruvate ratios evolution over time was evaluated separately for contralateral and TBI voxels using repeated measures (IBM SPSS Statistics v.23, *p<0.05, **p<0.01, ***p<0.005).

T2-weigthed images show CCI-induced TBI lesions in the right hemisphere (Fig.2A) and allowed proper positioning of the grid for HP ¹³C MRS. HP ¹³C spectra from the TBI voxels (red) show the increase of HP lactate at 12h, 24h and 7 days post-CCI and the return to baseline levels at 28d post-CCI. The increased of HP lactate was localized in the CCI-induced TBI hemisphere as shown in the HP-¹³C lactate heatmap at 12h post-TBI (Fig.2B). HP-lactate/pyruvate ratios, expressed as percentage change compared to baseline levels, are significantly increased in the TBI voxel (red) at 12h (p=0.005), 24h (p=0.046) and 7 days (p=0.009). In contrast, in the contralateral voxel (blue), no significant increase of HP lactate/pyruvate ratio could be observed at any time point (Fig.2C).

Importantly, IF analyses showed morphological changes of MPs (Iba-1, red), (Fig.2D). The ramified type, characteristic of resting microglia, was observed in the contralateral hemisphere while round/amoeboid-shaped MPs were observed in the TBI hemisphere. Furthermore, PDK1 (green) expression could only be detected in the TBI region where it co-localized with MPs (white arrows). Since PDK1 inhibits pyruvate dehydrogenase (PDH) and thus flux of pyruvate to the Krebs cycle, increased PDK1 in activated MPs provides a plausible mechanism for the increased lactate production observed in the TBI voxel, in line with a previous report ⁴. Further IF analyses are currently on-going to evaluate PDK1 expression at each time point.

In conclusion, our results demonstrate that ¹³C MRSI of HP pyruvate can detect increased lactate production in vivo in a preclinical model of TBI, and that this increase is linked to the presence of activated MPs. Importantly, this report is the first to demonstrate the use of a metabolic imaging method to monitor TBI-induced neuroinflammation. Because HP ¹³C MRSI is expanding rapidly, this study is of high significance for future clinical trials not only on TBI, but also all neurological diseases presenting an inflammatory component.