Hyperpolarized 13 C magnetic resonance spectroscopy detects toxin-induced neuroinflammation in mice

Abstract

Lipopolysaccharide (LPS) is a commonly used agent for induction of neuroinflammation in preclinical studies. Upon injection, LPS causes activation of microglia and astrocytes, whose metabolism alters to favor glycolysis. Assessing in vivo neuroinflammation and its modulation following therapy remains challenging, and new noninvasive methods allowing for longitudinal monitoring would be highly valuable. Hyperpolarized (HP) ¹³ C magnetic resonance spectroscopy (MRS) is a promising technique for assessing in vivo metabolism. In addition to applications in oncology, the most commonly used probe of [1-¹³ C] pyruvate has shown potential in assessing neuroinflammation-linked metabolism in mouse models of multiple sclerosis and traumatic brain injury. Here, we aimed to investigate LPS-induced neuroinflammatory changes using HP [1-¹³ C] pyruvate and HP ¹³ C urea. 2D chemical shift imaging following simultaneous intravenous injection of HP [1-¹³ C] pyruvate and HP ¹³ C urea was performed at baseline (day 0) and at days 3 and 7 post-intracranial injection of LPS (n = 6) or saline (n = 5). Immunofluorescence (IF) analyses were performed for Iba1 (resting and activated microglia/macrophages), GFAP (resting and reactive astrocytes) and CD68 (activated microglia/macrophages). A significant increase in HP [1-¹³ C] lactate production was observed at days 3 and 7 following injection, in the injected (ipsilateral) side of the LPS-treated mouse brain, but not in either the contralateral side or saline-injected animals. HP ¹³ C lactate/pyruvate ratio, without and with normalization to urea, was also significantly increased in the ipsilateral LPS-injected brain at 7 days compared with baseline. IF analyses showed a significant increase in CD68 and GFAP staining at 3 days, followed by increased numbers of Iba1 and GFAP positive cells at 7 days post-LPS injection. In conclusion, we can detect LPS-induced changes in the mouse brain using HP ¹³ C MRS, in alignment with increased numbers of microglia/macrophages and astrocytes. This study demonstrates that HP ¹³ C spectroscopy has substantial potential for providing noninvasive information on neuroinflammation.

Keywords: hyperpolarized 13C MRS; lipopolysaccharide; metabolism; neuroinflammation.

Figure 1

(A) MRS grid used for acquisition of HP ¹³C MRSI data overlaid on a T₂-weighted MR image, and voxels of interest post-voxel shift used for analysis (C=contralateral; I=ipsilateral). Stack of dynamic HP ¹³C spectra (grey), with summed spectrum at the rear (black), showing the resonances of HP [1-¹³C] pyruvate, HP [1-¹³C] lactate and HP ¹³C urea. (B) T₂-weighted MR images and summed HP ¹³C spectra from ipsilateral voxel at each timepoint, for both LPS (red) and saline (blue) animals.

Figure 2:

Heat maps showing HP [1-¹³C] lactate, HP [1-¹³C] pyruvate, and HP [1-¹³C] urea SNR following injection of HP [1-¹³C] pyruvate in an LPS-injected animal. Baseline, 3 day and 7 day data are shown.

Figure 3:

All data are shown for baseline (●), and at 3 days (■) and 7 days (▲) after surgery, in LPS and saline-treated groups. Further, all data were normalized to the contralateral side of the brain. (A) Data over time for individual metabolites assessed following injection of hyperpolarized [1-¹³C] pyruvate. HP [1-¹³C] lactate levels were significantly increased in LPS-treated animals at 7 days when compared to baseline levels and 3 day data (7 days: 164 ± 19% of baseline, p=0.0.0007; 136 ± 19% of 3 day data, p=0.018). (B) Ratios for ¹³C lactate/pyruvate, ¹³C pyruvate/urea and ¹³C lactate normalized to ¹³C pyruvate/urea. HP ¹³C lactate/pyruvate ratios were significantly increased in the LPS brains at 7 days when compared to baseline (150 ± 28% of baseline, p=0.0097). HP ¹³C lactate/(pyruvate/urea) ratios were significantly increased at 7 days compared to both baseline and 3 day data (7 days: 169 ± 23% of baseline p=0.026, 151 ± 23% of 3 day data, p=0.019). *p<0.05, **p<0.01, ***p<0.001

Figure 4:

Histological analysis of ipsi- and contra- lateral brain slices at each timepoint in the LPS-treated animals. Panels show Iba1, CD68 and GFAP staining examples (contralateral inset), with quantification of percentage coverage. (A) Iba1 staining was significantly increased in the ipsilateral side of LPS-treated animals at 7 days compared to baseline and 3 day levels, and at the 7 day timepoint compared to contralateral (7 days: 1096% of baseline, p<0.0001; 363% of 3 day data, p=0.0006, 835% of contralateral p=0.0001). (B) GFAP staining was significantly increased in the ipsilateral side of LPS-treated animals at 7 days compared to ipsilateral baseline and 3 day levels (7 days: 48550% of baseline, p<0.0001, 254% of 3 days, p=0.0002). Ipsilateral GFAP staining was also significantly higher at 3 days compared to baseline data (3 days: 19130% of baseline, p=0.022 (C) CD68 staining was elevated in the ipsilateral side at 3 days post-injection compared to the ipsilateral side at baseline and 7 days (3 day data: 1752% of baseline p=0.0008, 557% of 7 days p=0.003). At 3 days, CD68 was also significantly increased in the ipsilateral side as compared to contralateral (2769% of contralateral, p=0.005). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001