QSM is an imaging biomarker for chronic glial activation in multiple sclerosis lesions

Abstract

Background: Inflammation in chronic active lesions occurs behind a closed blood-brain barrier and cannot be detected with MRI. Activated microglia are highly enriched for iron and can be visualized with quantitative susceptibility mapping (QSM), an MRI technique used to delineate iron.

Objective: To characterize the histopathological correlates of different QSM hyperintensity patterns in MS lesions.

Methods: MS brain slabs were imaged with MRI and QSM, and processed for histology. Immunolabeled cells were quantified in the lesion rim, center, and adjacent normal-appearing white matter (NAWM). Iron⁺ myeloid cell densities at the rims were correlated with susceptibilities. Human-induced pluripotent stem cell (iPSC)-derived microglia were used to determine the effect of iron on the production of reactive oxygen species (ROS) and pro-inflammatory cytokines.

Results: QSM hyperintensity at the lesion perimeter correlated with activated iron⁺ myeloid cells in the rim and NAWM. Lesions with high punctate or homogenous QSM signal contained no or minimally activated iron^- myeloid cells. In vitro, iron accumulation was highest in M1-polarized human iPSC-derived microglia, but it did not enhance ROS or cytokine production.

Conclusion: A high QSM signal outlining the lesion rim but not punctate signal in the center is a biomarker for chronic inflammation in white matter lesions.

Figure 1

QSM signal accumulates at the lesion rim and correlates with the density of Perls⁺ cells. (A) Three lesions depicted on T2 and QSM. Left: QSM rim⁻ lesion with homogeneous susceptibility; middle: QSM rim⁺ lesion; right: QSM rim⁻ lesion with high susceptibility puncta. (B) Representative QSM rim⁺ lesion with a hyperintense rim that is located outside of the lesion area as defined by T2 FLAIR (left), and corresponding Perls stain (right). (C) Increased density of Perls⁺ cells correlates with increased susceptibility. Red dots represent rim⁺ lesions, black dots represent rim⁻ lesions. (D) Lesion with a central vein partially filled with fixed blood on Perls stain (left) visible on QSM (right).

Figure 2

Distribution of myeloid cells in rim+ and rim⁻ lesions. A representative rim+ lesion with (A) loss of myelin in the lesion center contains (B) a high density of myeloid cells at the lesion rim and (C) Perls⁺ cells with an activated morphology. A representative rim⁻ lesion with (D) complete loss of myelin contains (E) a thin layer of myeloid cells at the lesion rim but (F) lacks Perls⁺ cells. (G–H) Histological quantification. (I) Myeloid cells extend further into the NAWM of iron⁺ lesions as compared to iron⁻ lesions. Bars represent mean + SEM of n > 10 ROIs. Average linear density in Center, Rim, NAWM were compared between iron⁺ and iron⁻ lesions to determine statistical significance. MBP, myelin basic protein; *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 3

Distribution of activated myeloid cells iron⁺ and iron⁻ lesions. Iron⁺ lesions contain globoid and ramified myeloid cells that express pro‐inflammatory (A) iNOS, (B) Ferritin and anti‐inflammatory (C) CD206, (D) MerTK markers at higher density as compared to iron⁻ lesions. Bars represent mean + SEM of n > 10 ROIs. Total cells in Center, Rim, NAWM were compared between iron⁺ and iron⁻ lesions to determine statistical significance. iNOS, induction of nitric oxide synthase; *p < 0.05.

Figure 4

Iron uptake and production of reactive oxygen species in iPSC‐derived microglia. (A) M0, M1, and M2 polarized microglia cells (48 h, polarization with IFNγ/LPS (M1) and IL‐4 (M2)), stained with Perl’s staining after iron(III)sulfate exposure (B) Quantification of polarization‐dependent iron uptake by measurement of intracellular radioactive ⁵⁵Fe. (One‐way ANOVA (F = 21.58; p = 0.018); Tuckey–Kramer (M0 vs. M1 p = 0.0285; M0 vs M2 p = 0.0212; M1 vs. M2 p = 0.0015)). (C) CellROX measurements in unstimulated microglia cells, M1‐polarized (LPS/IFNγ), and alternatively stimulated cells (TNFα) after 48 h incubation with iron(III)sulfate. (D) mRNA expression levels and (E) protein expression of a panel of inflammation markers in M0 microglia and M1 polarized microglia cells after incubation with iron(III)sulfate (16 and 48 h).