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Metabolic imaging of brain inflammation using hyperpolarized 13C MRSI of pyruvate and urea in a mouse model of multiple sclerosis

Caroline Guglielmetti^1,2, Christian Cordano³, Chloe Najac⁴, Ari Green^3,5, and Myriam M. Chaumeil^1,2

¹Department of Physical Therapy and Rehabilitation Science, University of California San Francisco, San Francisco, CA, United States, ²Department of Radiology and Biomedical Imaging, University of California San Francisco, San Francisco, CA, United States, ³Division of Neuroimmunology and Glial Biology, Department of Neurology, University of California San Francisco, San Francisco, CA, United States, ⁴Department of Radiology, C.J. Gorter Center for High Field MRI, Leiden University Medical Center, Leiden, Netherlands, ⁵Department of Ophthalmology, University of California San Francisco, San Francisco, CA, United States

Synopsis

We used conventional MRI and hyperpolarized ¹³C magnetic resonance spectroscopy (HP ¹³C MRSI) quantitative imaging of pyruvate and urea to assess lesion pathology and the metabolic signature in a model of multiple sclerosis (MS). T₂sequences detected white matter lesions and gadolinium-enhanced MRI showed blood-brain-barrier breakdown. HP ¹³C MRSI revealed increased lactate production and lactate-to-pyruvate ratios while urea levels remained unchanged. This is consistent with macrophage/monocyte infiltration into the CNS found in the model. Altogether, these findings demonstrate that HP ¹³C MRSI has potential to monitor macrophage infiltration and innate immune activation in inflammatory diseases of the central nervous system.

Introduction

The combined cuprizone and experimental autoimmune encephalomyelitis (CPZ/EAE) is a recently developed model of multiple sclerosis (MS) that recapitulates key features of MS such as neurological symptoms, recruitment of T-cells into demyelinated brain lesions and increased number of innate immune cells^1,2. Several recent studies have shown that hyperpolarized ¹³C magnetic resonance spectroscopy imaging (HP ¹³C MRSI) of pyruvate can detect the presence of activated macrophages both in vitro and in vivo^3-9. In this study, our goal was to characterized the CPZ/EAE model using conventional MRI, HP ¹³C MRSI of pyruvate and urea, clinical evaluation of disease severity, evaluation of visual pathway conduction using visual evoked potential (VEP)¹⁰, and immunofluorescence (IF) analyses.

Methods

Animals and experimental setup: C57/BL6J mice were separated in three groups: 1-Control (n=3-5), 2-CPZ/SHAM (n=5-6) and 3-CPZ/EAE (n=5-6). Control received a normal chow while groups 2 and 3 were fed a CPZ diet (0.25%) for three weeks to induce brain demyelination/neuroinflammation, and were returned to a normal chow for five weeks. Groups 2 and 3 were SHAM-immunized or MOG_35-55-immunized, respectively. MRI, VEP and IF were performed at seven weeks post-induction of the disease, as described in Figure 1.A.

EAE scoring: Scoring of disease severity was performed as followed: 0) normal, 1) decreased tail tone, 2) hind limb weakness, 3) hind limb paralysis, 4) forelimbs weakness/paraplegia, 5) limbs paralysis.

MR acquisitions: MR acquisitions were performed on a 14.1T MR scanner. T₂-weighted images were acquired using: TE/TR=12/2000ms, thickness=0.5mm, NA=8, matrix=256x256, FOV=30x30mm². To evaluate blood brain barrier (BBB) integrity, T₁-weighted images post-gadolinium-DTPA injection were acquired: TE/TR=2/120ms, thickness=0.8mm, NA=10, matrix=256x256, FOV=20x20mm².

For ¹³C MRS, 2D dynamic CSI, 24μl [1-¹³C] pyruvate and 55μl ¹³C urea were co-polarized for ~1h in a Hypersense polarizer (Oxford Instruments), and rapidly dissolved in 4.5ml heated buffer (80mM NaOH in PBS). ¹³C data were acquired from the beginning of HP ¹³C pyruvate and urea injection: TE/TR=1.2/60ms; SW=2500Hz; 128points; 4sec resolution; FA=10deg; FOV=24x24 mm²; thickness=5mm.

MR analyses: Corpus callosum was delineated on T₂-weighted images and the average T₂ signals were normalized (nT₂) to T₂ from the cerebrospinal fluid.

For HP ¹³C MRSI, spectra were summed over time, HP ¹³C lactate, pyruvate, urea levels were calculated as the fit integrals.

VEP: Visual pathway conduction was examined by recording of flash VEP using an Espion Diagnosys setup. The time (in milliseconds) to the N1 (first negative deflection) was used to calculate VEP latency.

Immunofluorescence: IF analyses were performed for macrophages (Iba-1), CD3 T-cells and nuclei (Hoechst).

Statistical analyses: Statistical significance was evaluated using a One-Way ANOVA with post-hoc Tukey (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

Results

In this study, only mice from the CPZ/EAE group developed limb weakness and paralysis (Figure 2.A, p≤0.007). Similarly, only CPZ/EAE mice showed increased VEP latency at 7 weeks, indicating persistent injury to the visual pathway (Figure 2.B, p≤0.0001).

CPZ/EAE mice showed a hyper-intense corpus callosum on T₂-weighted MRI indicating demyelination and edema/inflammation (Figure 3.A, top row). Quantitative analyses revealed a significant increase of nT₂ values from the corpus callosum of CPZ/EAE compared to Control and CPZ/SHAM (Figure 3.B, p≤0.0355). Hyper-intense contrast seen on T₁-weighted Gadolinium-enhanced MRI clearly indicated leaky BBB in CPZ/EAE mice (Figure 3.A, bottom row).

CPZ/EAE mice displayed significantly higher level of HP ¹³C lactate as indicated on HP ¹³C spectra (Figure 4.A). Quantitative analyses revealed no changes in HP ¹³C pyruvate and urea levels between groups (Figure 4.B-D, p≤0.35 and p≤0.30, respectively). In contrast, HP ¹³C lactate levels, HP ¹³C lactate-to-pyruvate ratios, and HP ¹³C lactate-to-pyruvate ratios normalized to urea levels, were significantly increased in CPZ/EAE mice (Figure 4. E-F, p≤0.0049, p≤0.0095 and, p≤0.0068, respectively).

IF analyses revealed higher number of macrophages throughout the brain in CPZ/EAE mice (Figure 5.A-B, p≤0.0015) and infiltration of CD3 T-cells in close vicinity of blood vessels, beneath the hippocampus and thalamus (Figure 5.C).

Discussion

In conclusion, we showed that conventional T₂-weighted and gadolinium-enhanced imaging can detect demyelinated and inflammatory lesions in the CPZ/EAE model. HP ¹³C lactate production was increased in CPZ/EAE mice, in agreement with increased neuroinflammation levels. Interestingly, urea delivery did not increase despite a leaky BBB. Enzyme activity assays for pyruvate dehydrogenase and lactate dehydrogenase will be performed to further confirm the HP ¹³C MRSI result, and specific inhibitors may be used to further validate the origin of the HP ¹³C lactate signal. Future studies will include monitoring of response to anti-inflammatory therapies.

Acknowledgements

This work was supported by research grants: NIH R01NS102156, Cal-BRAIN 349087, NMSS research grant RG-1701-26630, Hilton Foundation – Marilyn Hilton Award for Innovation in MS Research #17319. Dana Foundation: The David Mahoney Neuroimaging program, NIH Hyperpolarized MRI Technology Resource Center #P41EB013598, fellowship from the NMSS (FG-1507-05297).

References

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Figures

Figure 1. Schematic overview of the experimental outline. Mice were separated in three groups. Control received a normal chow for the whole experimental period. Groups 2 and 3 received a CPZ diet for three weeks to induce brain demyelination/neuroinflammation, and were returned to a normal chow for five weeks. After two weeks of recovery (W5) mice from group 2 and 3 were SHAM-immunized or MOG_35-55-immunized, respectively. MRI, VEP and IF were performed at seven weeks (W7) post-induction of the disease.

Figure 2. (A) Assessment of disease severity (EAE score) showed that CPZ/EAE mice display limb paralysis (p≤0.007). (B) VEP latency was increased in CPZ/EAE mice compared to Control and CPZ/SHAM (p≤0.0001).

Figure 3. (A) T₂-weighted images revealed hyperintense corpus callosum in CPZ/EAE mice, indicating demyelination/neuroinflammation. (B) Quantitative analyses of the nT₂ values from the corpus callosum showed increased nT₂ values in the CPZ/EAE mice (p≤0.0355). (C) T₁-weighted images acquired post-injection of Gadolinium revealed hyperintense signal in CPZ/EAE mice in close vicinity of large blood vessels, indicating BBB breakdown.

Figure 4. (A) HP ¹³C MRS of pyruvate and urea revealed increased production of lactate in CPZ/EAE mice, as seen on the corresponding spectra. (B) HP ¹³C pyruvate and (C) HP ¹³C urea levels remain unchanged between groups despite alterations of the BBB in CPZ/EAE mice, as seen on the corresponding heatmaps (D). (E) HP ¹³C heatmaps of lactate and quantification of (F) HP ¹³C lactate levels, (G) HP ¹³C lactate-to-pyruvate ratios, and (H) HP ¹³C lactate-to-pyruvate ratios normalized to urea levels, were significantly increased in CPZ/EAE mice (p≤0.0049, p≤0.0095 and, p≤0.0068, respectively).

Figure 5. (A) IF analyses demonstrated higher number of macrophages (Iba-1, red) throughout the brain in CPZ/EAE mice. (B) Quantitative analyses of macrophages (Iba-1) present in the corpus callosum of CPZ/EAE mice was significantly higher than in Control and CPZ/SHAM mice (p≤0.0015).(C) IF performed at the level of the hippocampus and thalamus revealed a high number of cells (Hoechst, blue) including macrophages (Iba-1, red) and CD3 T-cells (CD3, green).

Proc. Intl. Soc. Mag. Reson. Med. 27 (2019)

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