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Hyperpolarized 13C MRSI of pyruvate and urea can detect immunomodulatory responses to dimethyl fumarate therapy in a model of multiple sclerosis1Department 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, 3Department 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
Synopsis
We used hyperpolarized 13C magnetic resonance spectroscopic imaging (HP 13C MRSI) and T1-MRI to assess dimethyl fumarate (DMF) response in a model of multiple sclerosis (MS). Gadolinium-enhanced T1-MRI showed blood-brain-barrier breakdown, regardless of DMF treatment. In contrast, DMF therapy prevented an increase compared to untreated MS animals in HP 13C lactate, HP 13C lactate-to-pyruvate and HP 13C lactate-to-(pyruvate/urea) ratios. HP 13C MRSI findings were further correlated to pyruvate dehydrogenase activity and pro-inflammatory macrophages. Altogether, we demonstrated that HP 13C MRSI has potential to monitor the effect of immunomodulatory therapies within the central nervous system.
Introduction
Hyperpolarized 13C magnetic resonance spectroscopy/spectroscopic imaging (HP 13C MRS/I) has demonstrated capacity for detecting pro-inflammatory cells in vitro and in various organs, including lungs, liver, heart, joints and brain1-8. Pro-inflammatory innate immune cells play a crucial role in multiple sclerosis (MS) pathophysiology9,10. Because of its clinical effect on disease activity and related immunomodulatory properties, dimethyl fumarate (DMF) is used as a treatment for MS. In this study, we investigated whether HP 13C MRSI is sensitive to treatment with DMF. To do so, we measured the conversion of HP 13C pyruvate to lactate as a proxy for the detection of pro-inflammatory cells5-7. As the blood brain barrier (BBB) is compromised in MS, we co-injected HP 13C pyruvate with HP 13C urea, which served as a perfusion imaging probe. The imaging findings were validated and correlated to clinical symptoms, enzymatic activity and immunofluorescence.Methods
Animals and experimental design: C57/BL6J mice were separated in three groups: 1-Control (n=4-7), 2-Cuprizone and Experimental Autoimmune Encephalomyelitis11,12 (CPZ-EAE) (n=5-9) and 3-CPZ-EAE+DMF (n=6-9). Control received a normal chow. Groups 2 and 3 received CPZ diet (0.25%) for three weeks (W0-W3). At W5, groups 2 and 3 were MOG35-55-immunized, and group 3 received DMF (100 mg/kg/day) for two weeks. MRI, enzymatic assays and immunofluorescence were performed at W7 (Figure 1.A).EAE scoring: Disease severity was scored as: 0) normal, 1) decreased tail tone, 2) hind limb weakness, 3) hind limb paralysis, 4) forelimbs weakness/paraplegia, 5) limbs paralysis.
MR acquisitions and analyses: MR acquisitions were performed on a 14.1T Agilent MR scanner. To evaluate BBB integrity, T1-weighted images were acquired prior and five minutes post intravenous gadolinium-DTPA injection (1 mmol/kg). T1-weighted maps were created using: (T1post-T1pre)/T1pre*100. For 13C MRS, 24μl [1-13C] pyruvate and 55μl 13C urea were co-polarized for ~1h in a Hypersense polarizer (Oxford Instruments) and dissolved in 4.5mL buffer (80mM NaOH in PBS). 2D dynamic CSI 13C data were acquired from the beginning of injection. 13C spectra were summed over time and HP pyruvate, lactate and urea levels were calculated as the fit integrals. MR acquisition parameters are summarized in Figure 1.B.
Immunofluorescence: Immunofluorescence analyses were performed for BBB integrity (Fibrinogen), resting/activated macrophages (Iba1), pro-inflammatory (CD68) and anti-inflammatory (Arginase1) macrophages.
Enzymatic assays: Pyruvate dehydrogenase (PDH) and lactate dehydrogenase (LDH) activity were measured by spectrophotometric assays.
Statistical analyses: Statistical significance was evaluated using a One-Way ANOVA with post-hoc Tukey. Correlations analyses were performed using Pearson coefficient correlation or linear regression (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).
Results
CPZ-EAE mice showed limb weakness and paralysis at W7 (EAE score = 3.6±0.7) while mice treated with DMF showed significantly less severe symptoms (EAE score = 1.1±0.8, p<0.0001).Following injection of HP 13C pyruvate and urea, CPZ-EAE mice display an increased 13C lactate production compared to control, whereas CPZ-EAE mice treated with DMF showed a 13C lactate production comparable to control (Figure 2.A). HP 13C pyruvate and urea were not significantly different between groups; in contrast, HP 13C lactate was significantly increased in CPZ-EAE mice compared to control and CPZ-EAE+DMF (Figure 2, Table). HP 13C lactate-to-pyruvate and HP 13C lactate-to-(pyruvate/urea) ratios were significantly increased in CPZ-EAE mice compared to control (Figure 2.B-C, +225±63%, p=0.0043, and +248±73%, p=0.0015, respectively), while DMF treatment prevented this increase (-154±38% decrease from CPZ-EAE, p=0.0374 and -225±24%, p=0.0011, respectively). Modulations of the HP 13C ratios could be observed in the whole brain (Figure 2.D). PDH enzymatic activity was strongly decreased in CPZ-EAE compared to control (Figure 2.E, p<0.0001) and DMF partially prevented this decrease (p=0.0034), providing an explanation for the increased lactate production. LDH activity remained unchanged (Figure 2.F).
A leaky BBB could be observed in both CPZ-EAE and CPZ-EAE+DMF mice, as indicated by increased T1 contrast in the thalamic and cortical regions following gadolinium-DTPA injection (Figure 3.A-B, p=0.0282 and p=0.0497, respectively) and fibrinogen deposition (Figure 4.A-B, p=0.0164 and p=0.0159, respectively), together with an increased level of macrophages (Figure 4.C, p=0.0007 and p=0.0029, respectively). CPZ-EAE mice showed high levels of pro-inflammatory macrophages throughout the brain (Figure 4.D, p=0.0018), while mice DMF-treated showed reduced levels of pro-inflammatory cells (-469±102% decrease from CPZ-EAE, p=0.0062). In addition, the number of anti-inflammatory macrophages was increased following DMF therapy (Figure 4.E, +347±73% increase from CPZ-EAE, p=0.0270), confirming DMF's effect on innate activity.
Correlations analyses revealed strong associations between HP 13C lactate-to-pyruvate/HP 13C lactate-to-(pyruvate/urea) and PDH activity (Figure 5, p<0.0001 and p=0.0006), pro-inflammatory CD68 macrophages (p=0.0004 and p=0.0003), as well as EAE score (p=0.0014 and p=0.0023) and total macrophage levels (p=0.0032 and p=0.042). Only HP 13C lactate-to-pyruvate was associated with BBB integrity as measured by fibrinogen deposition (p=0.0393).
Discussion
We showed that DMF treatment prevented the increase of HP 13C lactate-to-pyruvate and HP 13C lactate-to-(pyruvate/urea) ratios and the decrease of PDH activity observed in the CPZ-EAE MS model, while shifting the balance from pro-inflammatory towards anti-inflammatory macrophages. Despite a leaky BBB, delivery of HP 13C pyruvate or urea remained unchanged between groups. Altogether our findings demonstrated that HP 13C MRSI is sensitive to DMF therapy in an MS model, highlighting the potential of this method to non-invasively monitor the effects of immunomodulatory treatments in the central nervous system.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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