Hyperpolarized 13C MR metabolic imaging can detect neuroinflammation in vivo in a multiple sclerosis murine model

Abstract

Proinflammatory mononuclear phagocytes (MPs) play a crucial role in the progression of multiple sclerosis (MS) and other neurodegenerative diseases. Despite advances in neuroimaging, there are currently limited available methods enabling noninvasive detection of MPs in vivo. Interestingly, upon activation and subsequent differentiation toward a proinflammatory phenotype MPs undergo metabolic reprogramming that results in increased glycolysis and production of lactate. Hyperpolarized (HP) ¹³C magnetic resonance spectroscopic imaging (MRSI) is a clinically translatable imaging method that allows noninvasive monitoring of metabolic pathways in real time. This method has proven highly useful to monitor the Warburg effect in cancer, through MR detection of increased HP [1-¹³C]pyruvate-to-lactate conversion. However, to date, this method has never been applied to the study of neuroinflammation. Here, we questioned the potential of ¹³C MRSI of HP [1-¹³C]pyruvate to monitor the presence of neuroinflammatory lesions in vivo in the cuprizone mouse model of MS. First, we demonstrated that ¹³C MRSI could detect a significant increase in HP [1-¹³C]pyruvate-to-lactate conversion, which was associated with a high density of proinflammatory MPs. We further demonstrated that the increase in HP [1-¹³C]lactate was likely mediated by pyruvate dehydrogenase kinase 1 up-regulation in activated MPs, resulting in regional pyruvate dehydrogenase inhibition. Altogether, our results demonstrate a potential for ¹³C MRSI of HP [1-¹³C]pyruvate as a neuroimaging method for assessment of inflammatory lesions. This approach could prove useful not only in MS but also in other neurological diseases presenting inflammatory components.

Keywords: hyperpolarized 13C MR spectroscopy; macrophages; metabolism; multiple sclerosis; neuroinflammation.

Fig. 1.

Histological and MRI characterization of CPZ mice. (A) Myelin (MBP), microglia/macrophages (Iba-1, MPs), oligodendrocyte progenitor cells (OPCs, PDGFR-α), and astrocytes (GFAP) staining of the corpus callosum (dashed lines) prior to (W0) and following CPZ diet (W4, W6), and after recovery (W6 + W6 recovery). (Scale bar: 100 µm.) (B) Immunofluorescence staining illustrates the massive cell infiltration of activated MPs (Iba-1; nuclear staining Hoechst) in the corpus callosum (white arrows) at W4 of CPZ compared with CTRL. (Magnification: 10×; scaling per pixel, 0.645 μm × 0.645 μm.) (C) Quantitative analyses of the corpus callosum confirm demyelination (MBP, P < 0.0001) and maximal microgliosis (Iba-1, P < 0.0001) after W4 of CPZ. Remyelination and decreased MP levels are observed following W6 of CPZ diet (P = 0.0285 and P < 0.0001, respectively). Myelin and MP levels return to CTRL levels at W6 + W6 recovery. OPCs (PDGFR-α) accumulate at W4 of CPZ (P = 0.0299) and return to CTRL levels at W6 + W6 recovery (P = 0.0479). Astrocyte (GFAP) staining quantification shows highest astrogliosis after W6 of CPZ (P < 0.0001), which persists after the recovery period (P < 0.0001) (n = 3–5 mice per group). All values are reported as mean ± SD. (D) T₂-weighted MR images show hyperintensity in the corpus callosum at W4 and W6 of CPZ diet (white arrows). Corresponding nT₂w values increased at W4 and W6 of CPZ (P ≤ 0.0003), in line with demyelination and neuroinflammation at these time points. At W6 + W6 recovery, hypointense contrast can be observed in the corpus callosum, resulting in a decrease of nT₂w toward CTRL values (P = 0.0011), indicating remyelination and decreased neuroinflammation (n = 5–7 mice per group). In the CTRL group, the nT₂w values decreased over time, reflecting myelination of the corpus callosum in young adult mice. All values are reported as mean ± SD (one-way ANOVA and two-Way ANOVA, Tukey HSD post hoc test *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; unpaired t test ^##P < 0.01; repeated measures ANOVA ^†P < 0.05). n.s., not significant. A.U., arbitrary unit.

Fig. S1.

Experimental outline of the study and representative HP ¹³C dataset. (A) CTRL and 0.2% CPZ animals were imaged using T₂-weighted MRI and ¹³C MRSI of HP [1-¹³C]pyruvate at four distinct time points: week 0, 4, 6, and after a 6-wk period of standard chow diet to allow recovery (W0, W4, W6, and W6 + W6 recovery). A subset of animals was killed at each time point for immunofluorescence analysis and enzyme activity assays. (B) T₂-weighted MR image of a mouse head is overlaid with the grid used for HP ¹³C MR acquisition. The region of interest (corpus callosum) is highlighted in red (white box). Corresponding stack plot of HP ¹³C spectra show HP [1-¹³C]pyruvate delivery and subsequent HP [1-¹³C]lactate production over time in the voxel of interest [3-s temporal resolution, 16 time points (TP)]. All dynamic spectra (shown in black) are summed (shown in red) and integrals of HP [1-¹³C]pyruvate and HP [1-¹³C]lactate calculated for each imaging session and each animal.

Fig. 2.

Increased production of HP [1-¹³C]lactate in CPZ-induced lesions. (A) HP ¹³C spectra from the corpus callosum (white voxels) show an increased HP [1-¹³C]lactate production after W4 of CPZ diet. (B) HP [1-¹³C]pyruvate delivery is unchanged between CTRL and CPZ at any time point of interest. (C) When comparing CPZ and CTRL groups, the HP [1-¹³C]lactate-to-pyruvate ratios are significantly increased after W4 of CPZ diet (315 ± 338%, P = 0.0006). (D) Longitudinal analyses of the HP [1-¹³C]lactate-to-pyruvate ratios between W0 (blue), W4 CPZ (red), W6 CPZ (green), and W6 + W6 recovery (purple) show a significant increase at W4 (96 ± 74% of W0, P = 0.0002) followed by a decrease at W6 and W6 + W6 recovery (P = 0.0194 and P = 0.0346, respectively). The two animals that were not imaged at W6 + W6 were excluded from the statistical analysis between W4 and W6 + W6. HP [1-¹³C]lactate-to-pyruvate ratios of the CTRL mice (open circles) did not show any significant differences over time (n = 5–7 mice per group). All values are reported as mean ± SD (two-way ANOVA, Tukey HSD post hoc test *P < 0.05, ***P < 0.001, ****P < 0.0001; unpaired t test ^#P < 0.05). A.U., arbitrary unit; Lac, lactate; Pyr, pyruvate.

Fig. S2.

Contrast-enhanced MRI following CPZ diet. (A) Representative T₁-weighted MR images of a CTRL mouse and a mouse at W4 CPZ, prior to (pre-contrast) and after (post-contrast) injection of a gadolinium-based contrast agent (arrow indicates the time of contrast agent injection), showing characteristic enhancement of surrounding tissues and blood vessels on postcontrast images. Time courses of signal intensity (B) for the corpus callosum and the thalamus, showing an increase in both regions following contrast agent injection in CTRL and W4 CPZ mice. Quantitative analyses of the area under the curve (C), slope (D), and relative signal enhancement (E) showed no significant differences between CTRL and W4 CPZ mice (n = 3 mice per group). All values are reported as mean ± SD, except D, which displays mean ± SEM. AU, arbitrary unit.

Fig. 3.

Increased HP [1-¹³C]lactate-to-pyruvate ratio is restricted to the inflamed and demyelinated corpus callosum. (A) HP [1-¹³C]lactate-to-pyruvate ratio heat maps display an increased HP [1-¹³C]lactate production after W4 of CPZ diet in the corpus callosum only. (B) T₂-weighted image overlaid with the grid used for HP ¹³C MR acquisition is shown for a W4 CPZ mouse. The corresponding sum of dynamic spectra is presented. Increased HP [1-¹³C]lactate can be observed in the voxel containing the corpus callosum (red) but not in a voxel containing the thalamic region (dark blue) or in a nonbrain voxel located beneath the brain (neck voxel, light blue). (C and D) HP [1-¹³C]pyruvate delivery as well as (E and F) HP [1-¹³C]lactate-to-pyruvate ratios were not significantly different between CTRL and CPZ groups over time in the thalamic and neck region (n = 5–7 mice per group; C and E thalamus and D and F neck). All values are reported as mean ± SD. A.U., arbitrary unit.

Fig. 4.

Increased HP [1-¹³C]lactate production is associated with up-regulation of PDK1 in activated MPs and decreased PDH activity in the corpus callosum. (A) Immunofluorescence staining for PDK1 (green) and MPs (Iba-1, red) reveal overexpression of PDK1 in MPs after W4 of CPZ diet (yellow). Quantitative analyses confirmed the significant up-regulation of PDK1 after W4 of CPZ (P < 0.0001), (n = 3 mice per time point). (Scale bar: 100 µm.) (B) Confocal microscopy confirmed cellular colocalization (yellow, arrows) of PDK1 (green) in MPs (Iba-1, red) at W4 of CPZ diet. In contrast, astrocytes (GFAP, red, arrow) and OPCs (PDGFR-α, red, arrow) staining did not colocalize with PDK1 staining (green) at W4 of CPZ diet, as shown by confocal microscopy. (Scale bar: 10 µm.) (C) Quantitative analyses revealed a significant decrease of PDH enzyme activity at W4 of CPZ diet (P = 0.0026) and (D) no significant change in LDH-A enzyme activity (reported as V_max). (E) Serum lactate levels were significantly increased at W4 and W6 of CPZ diet (161 ± 62% W4 P = 0.0014; 166 ± 48% W6 P = 0.0007) and returned to CTRL levels by the end of the recovery period (P = 0.0024), (n = 4–6 mice per time point). All values are reported as mean ± SD (one-way ANOVA, Tukey HSD post hoc test **P < 0.01, ***P < 0.001).

Fig. S3.

Immunofluorescence staining of the corpus callosum, thalamus, and cortex following CPZ diet and evaluation of PDH activity in the thalamus. (A) Representative immunofluorescence images of MPs (Iba-1, red) and PDK1 (green) in the thalamus and cortex showing no expression of PDK1, and hence no colocalization with Iba-1 at any time point of interest (n = 3 mice per group). (Scale bar: 100 µm.) (B) Representative immunofluorescence staining of astrocytes (GFAP, red) and OPCs (PDGFR-α, red) showing no colocalization with PDK1 staining (green) in the corpus callosum at any time points of interest (n = 3 mice per group). (Scale bar: 100 µm.) (C) Quantitative analyses of PDH enzyme activity in the thalamus revealed no significant changes following CPZ diet (n = 4–7 mice per group). (D) Immunofluorescence staining for LDH-A and nuclear staining (Hoechst) show that LDH-A is not detected in the corpus callosum following W4 of CPZ diet. LDH-A expression was found in the CTRL area CA-1 of the hippocampus (n = 3 mice per group). (Magnification: 20×.)

Fig. 5.

HP [1-¹³C]lactate levels are not increased in the corpus callosum of CX₃CR1^GFP/GFP transgenic mice harboring microglial activation deficiency, suggesting a central role of MPs to the detected HP [1-¹³C]lactate signal. (A) T₂-weighted MRI of CX₃CR1^GFP/GFP mice show similar hypointense contrast of the corpus callosum regardless of diet (CTRL or W4 CPZ). nT₂w were significantly decreased at W4 in CPZ and CTRL CX₃CR1^GFP/GFP, reflecting normal myelination (P ≤ 0.0171). Immunofluorescence images of CX₃CR1^GFP/GFP microglia show no evident microgliosis in the corpus callosum of CX₃CR1^GFP/GFP mice despite the CPZ diet. (Scale bar: 100 µm.) (B) PDH and (C) LDH-A activities from the corpus callosum were not significantly different between CPZ and CTRL groups. (D) Serum lactate levels were significantly increased following W4 CPZ, in line with a systemic toxic effect of CPZ diet (173 ± 79%, P = 0.0079). (E) T₂-weighted image overlaid with the HP ¹³C grid is shown for a W4 CPZ CX₃CR1^GFP/GFP mouse. HP ¹³C spectra for CTRL and W4 CPZ CX₃CR1^GFP/GFP mice show no evident differences between CTRL and CPZ groups in any studied voxel (corpus callosum red; thalamic region dark blue; neck voxel light blue). HP [1-¹³C]lactate-to-pyruvate ratios for (F) the corpus callosum, (G) the thalamic area, and (H) the neck were not significantly different between CTRL and CPZ groups at any time points (n = 5–7 mice per group). All values are reported as mean ± SD (two-way ANOVA, Tukey HSD post hoc test *P < 0.05; unpaired t test ^##P < 0.01). A.U., arbitrary unit; Lac, lactate; Pyr, pyruvate.

Fig. S4.

Immunofluorescence staining and HP [1-¹³C]pyruvate delivery in CX₃CR1^GFP/GFP mice following W4 of CPZ diet. (A) Representative immunofluorescence staining for cell infiltration (Hoechst, blue), myelin (MBP, red), astrocytes (GFAP, red), and MPs (Iba-1, red) shows no evident changes between CX₃CR1^GFP/GFP mice that received a CTRL chow or W4 of CPZ diet. (Scale bar: 100 µm.) HP [1-¹³C]pyruvate levels in CX₃CR1^GFP/GFP were not significantly different between CTRL and CPZ groups over time in (B) the corpus callosum, (C) the thalamic area, and (D) the neck region (n = 6 mice per group). All values are reported as mean ± SD. A.U., arbitrary unit.

Fig. 6.

Detection of PDK1-expressing MPs after EAE induction. (A) Immunofluorescence staining for MPs (Iba-1, red) shows increased number of MPs and expression of PDK1 (green) in the spinal cord 30 d after EAE induction compared with age- and sex-matched CTRL. (B and C) Quantitative analyses reveal that 45 ± 8% of MPs present in the lesions coexpress Iba-1 and PDK1 (yellow) in the spinal cord, 28 ± 8% in the brainstem, and 26 ± 7% in the cerebellum, whereas the level of PDK1 in the CTRL group was below detection. All values are reported as mean ± SD (n = 3 mice).