Quantitative susceptibility mapping identifies inflammation in a subset of chronic multiple sclerosis lesions

Abstract

Chronic active multiple sclerosis lesions, characterized by a hyperintense rim of iron-enriched, activated microglia and macrophages, have been linked to greater tissue damage. Post-mortem studies have determined that chronic active lesions are primarily related to the later stages of multiple sclerosis; however, the occurrence of these lesions, and their relationship to earlier disease stages may be greatly underestimated. Detection of chronic active lesions across the patient spectrum of multiple sclerosis requires a validated imaging tool to accurately identify lesions with persistent inflammation. Quantitative susceptibility mapping provides efficient in vivo quantification of susceptibility changes related to iron deposition and the potential to identify lesions harbouring iron-laden inflammatory cells. The PET tracer 11C-PK11195 targets the translocator protein expressed by activated microglia and infiltrating macrophages. Accordingly, this study aimed to validate that lesions with a hyperintense rim on quantitative susceptibility mapping from both relapsing and progressive patients demonstrate a higher level of innate immune activation as measured on 11C-PK11195 PET. Thirty patients were enrolled in this study, 24 patients had relapsing remitting multiple sclerosis, six had progressive multiple sclerosis, and all patients had concomitant MRI with a gradient echo sequence and PET with 11C-PK11195. A total of 406 chronic lesions were detected, and 43 chronic lesions with a hyperintense rim on quantitative susceptibility mapping were identified as rim+ lesions. Susceptibility (relative to CSF) was higher in rim+ (2.42 ± 17.45 ppb) compared to rim- lesions (-14.6 ± 19.3 ppb, P < 0.0001). Among rim+ lesions, susceptibility within the rim (20.04 ± 14.28 ppb) was significantly higher compared to the core (-5.49 ± 14.44 ppb, P < 0.0001), consistent with the presence of iron. In a mixed-effects model, 11C-PK11195 uptake, representing activated microglia/macrophages, was higher in rim+ lesions compared to rim- lesions (P = 0.015). Validating our in vivo imaging results, multiple sclerosis brain slabs were imaged with quantitative susceptibility mapping and processed for immunohistochemistry. These results showed a positive translocator protein signal throughout the expansive hyperintense border of rim+ lesions, which co-localized with iron containing CD68+ microglia and macrophages. In conclusion, this study provides evidence that suggests that a hyperintense rim on quantitative susceptibility measure within a chronic lesion is a correlate for persistent inflammatory activity and that these lesions can be identified in the relapsing patients. Utilizing quantitative susceptibility measure to differentiate chronic multiple sclerosis lesion subtypes, especially chronic active lesions, would provide a method to assess the impact of these lesions on disease progression.

Figures

Figure 1

Demonstration of chronic QSM rim+ and rim− lesions on T2 and QSM.Top: QSM rim+ lesion on MRI T2 sequence (A) and QSM (B). Bottom: Example of QSM rim− lesion on T2 (C) and QSM (D).

Figure 2

Comparison of PK11195-PET uptake in rim− and rim+ lesions. (A) Box plots of QSM rim− and rim+ lesions demonstrate higher VT ratio (VTr) value in rim+ lesions compared to rim− lesions. (B) Confidence intervals derived from the mixed effects model, controlling for patient age and volume demonstrate a clear distinction in VTr value among rim− versus rim+ lesions (P < 0.015).

Figure 3

Demonstration of chronic QSM rim+ and rim− lesions on QSM and PK11195-PET.Top: QSM rim+ lesion on QSM (A and C) and PK11195-PET (B and D). Bottom: QSM rim− lesion on QSM (C and G) and PK11195-PET (D and H).

Figure 4

Expression of TSPO in iron-positive and iron-negative chronic active white matter lesions. (A) White matter lesion with a broad rim of increased susceptibility on MRI (red circle identifies rim+ lesion, arrow identifies lesion rim). (B) Histological sections of this lesion show iron-positive cells at the lesion rim, identified with Perls’ staining (brown, counterstained with neutral red). (C and D) These cells are predominantly CD68+ microglia and macrophages that express TSPO, as seen in the high magnification inset (both brown, counterstained with haematoxylin). (E) White matter lesion with isointense susceptibility (blue circle identifies rim− lesion, arrow identifies lesion border). (F) There are no iron-positive cells present at the narrow lesion border and (G) only few CD68+ microglial cells. (H) Microglia at the lesion border also express TSPO (inset). Large panel scale bars = 100 µm. Inset scale bars = 20 µm.

Figure 5

Expression of CD68+ microglia cells and GFAP+ astrocytes within TSPO+ cells. (A) Composite confocal microscopy image at the rim of an iron-positive, chronic active white matter lesion, showing fluorescent TSPO immunoreactivity (red, A, B, E and F) in astrocytes and microglia/macrophages labelled with fluorescent antibodies against glial fibrillary acidic protein (GFAP, green, A, C and E) and CD68 (cyan, A, D and F), respectively. E is a merge of B and C, and F is a merge of B and D. (G) Percentage of TSPO+ cells that were either CD68+ (58.26%), GFAP+ (26.61%) or negative (15.13%) for both markers. A total of 218 TSPO+ cells were counted. (H) Densitometric quantification of average TSPO expression in GFAP+ and CD68+ cells. Data in (H) represent means ± SD. Scale bar = 20 µm in A–F.