CEST MRI and MALDI imaging reveal metabolic alterations in the cervical lymph nodes of EAE mice

Abstract

Background: Multiple sclerosis (MS) is a neurodegenerative disease, wherein aberrant immune cells target myelin-ensheathed nerves. Conventional magnetic resonance imaging (MRI) can be performed to monitor damage to the central nervous system that results from previous inflammation; however, these imaging biomarkers are not necessarily indicative of active, progressive stages of the disease. The immune cells responsible for MS are first activated and sensitized to myelin in lymph nodes (LNs). Here, we present a new strategy for monitoring active disease activity in MS, chemical exchange saturation transfer (CEST) MRI of LNs.

Methods and results: We studied the potential utility of conventional (T2-weighted) and CEST MRI to monitor changes in these LNs during disease progression in an experimental autoimmune encephalomyelitis (EAE) model. We found CEST signal changes corresponded temporally with disease activity. CEST signals at the 3.2 ppm frequency during the active stage of EAE correlated significantly with the cellular (flow cytometry) and metabolic (mass spectrometry imaging) composition of the LNs, as well as immune cell infiltration into brain and spinal cord tissue. Correlating primary metabolites as identified by matrix-assisted laser desorption/ionization (MALDI) imaging included alanine, lactate, leucine, malate, and phenylalanine.

Conclusions: Taken together, we demonstrate the utility of CEST MRI signal changes in superficial cervical LNs as a complementary imaging biomarker for monitoring disease activity in MS. CEST MRI biomarkers corresponded to disease activity, correlated with immune activation (surface markers, antigen-stimulated proliferation), and correlated with LN metabolite levels.

Keywords: CEST MRI; Lymph nodes; Multiple sclerosis; Neuroinflammation.

Fig. 1

Altered immune cell profiles in EAE-induced mice at 14 DPI. Comparison of immune cell composition in (A–C) CNS-draining, superficial cervical LNs and (D–F) in CNS tissue of EAE (n = 5) and naïve (n = 3) mice. Presence (%) of established activation markers were assessed for (A, D) T cells gated using CD3 + , and (B, E) myeloid cells and (C, F) dendritic cells gated using CD45 + in LNs and CNS tissue. E = EAE mice, N = naïve mice. *p < 0.05, **p < 0.01, ***p < 0.001 using ANOVA with Bonferroni post-hoc

Fig. 2

MRI of CNS-draining, superficial cervical LNs during EAE progression. A Disability score during the course of EAE. Arrows highlight days MRI was performed. B Visualization of the LNs using T2-weighted (T2-w) MRI. Quantification of (C) T2-w MRI and (D, E) CEST MRI signal intensity as disability progressed. Frequency ranges (± 0.2 ppm) whose average MTR_asym-based CEST signals in EAE-induced mice (n = 8) and not in control mice (n = 4) significantly (p < 0.05) differed include (F) 0.4–6.0, (G) 1.6, (H) 3.2, and (I) 5.2 ppm. cLN cervical LN. *p < 0.05, **p < 0.01, ***p < 0.001 using ANOVA with Bonferroni post-hoc

Fig. 3

MRI of CNS-draining, superficial cervical LNs at 14 DPI. Visualization of MR signal in (A, C) EAE-induced and (B, D) control (no MOG_35-55) mice. A, B Overlay of average MTR_asym-based CEST map (0.4–6.0 ppm) of LNs on T2-w image for anatomical reference. C, D Isolated T2-w and average MTR_asym-based CEST maps at frequency ranges (± 0.2 ppm) with significant (p < 0.05 using ANOVA with Bonferroni post-hoc) alterations of signal in EAE-induced mice and not in control mice: 0.4–6.0, 1.6, 3.2, and 5.2 ppm

Fig. 4

Spearman's rank correlation of MTR_asym signal in EAE-induced mice (13–14 DPI) to immune cell composition (14–15 DPI). Comparison to (A) immune cell activity (14–15 DPI; n = 5) and to (B) paralysis severity (29 DPI; n = 8). Comparison to (C–F) immune cell (T cell, myeloid cell; 15 DPI) profile (%) in (C, D) CNS tissue (n = 6) and in (E, F) CNS-draining, superficial cervical LNs (n = 5). G Comparison to metabolites in CNS-draining, superficial cervical LNs (14–15 DPI; n = 5). *p < 0.05

Fig. 5

MALDI imaging of the top 5 most significantly altered metabolites that correlate with MTR_asym signal in EAE-induced mice at 13–14 DPI. Visualization of alanine, lactate, leucine, malate and phenylalanine presence in representative cLNs of (top row) control and (middle row) EAE mice. (Bottom row) Average (n = 5) signal intensity of these metabolites at their characteristic mass-to-charge ratio (m/z). Optical imaging is shown to highlight the location of the cLNs. H&E staining is shown to highlight intra-nodal regions (medulla and cortex). Arrows highlight prominent examples of these regional differences