Contribution of Tissue Inflammation and Blood-Brain Barrier Disruption to Brain Softening in a Mouse Model of Multiple Sclerosis

Abstract

Neuroinflammatory processes occurring during multiple sclerosis cause disseminated softening of brain tissue, as quantified by in vivo magnetic resonance elastography (MRE). However, inflammation-mediated tissue alterations underlying the mechanical integrity of the brain remain unclear. We previously showed that blood-brain barrier (BBB) disruption visualized by MRI using gadolinium-based contrast agent (GBCA) does not correlate with tissue softening in active experimental autoimmune encephalomyelitis (EAE). However, it is unknown how confined BBB changes and other inflammatory processes may determine local elasticity changes. Therefore, we aim to elucidate which inflammatory hallmarks are determinant for local viscoelastic changes observed in EAE brains. Hence, novel multifrequency MRE was applied in combination with GBCA-based MRI or very small superparamagnetic iron oxide particles (VSOPs) in female SJL mice with induced adoptive transfer EAE (n = 21). VSOPs were doped with europium (Eu-VSOPs) to facilitate the post-mortem analysis. Accumulation of Eu-VSOPs, which was previously demonstrated to be sensitive to immune cell infiltration and ECM remodeling, was also found to be independent of GBCA enhancement. Following registration to a reference brain atlas, viscoelastic properties of the whole brain and areas visualized by either Gd or VSOP were quantified. MRE revealed marked disseminated softening across the whole brain in mice with established EAE (baseline: 3.1 ± 0.1 m/s vs. EAE: 2.9 ± 0.2 m/s, p < 0.0001). A similar degree of softening was observed in sites of GBCA enhancement i.e., mainly within cerebral cortex and brain stem (baseline: 3.3 ± 0.4 m/s vs. EAE: 3.0 ± 0.5 m/s, p = 0.018). However, locations in which only Eu-VSOP accumulated, mainly in fiber tracts (baseline: 3.0 ± 0.4 m/s vs. EAE: 2.6 ± 0.5 m/s, p = 0.023), softening was more pronounced when compared to non-hypointense areas (percent change of stiffness for Eu-VSOP accumulation: −16.81 ± 16.49% vs. for non-hypointense regions: −5.85 ± 3.81%, p = 0.048). Our findings suggest that multifrequency MRE is sensitive to differentiate between local inflammatory processes with a strong immune cell infiltrate that lead to VSOP accumulation, from disseminated inflammation and BBB leakage visualized by GBCA. These pathological events visualized by Eu-VSOP MRI and MRE may include gliosis, macrophage infiltration, alterations of endothelial matrix components, and/or extracellular matrix remodeling. MRE may therefore represent a promising imaging tool for non-invasive clinical assessment of different pathological aspects of neuroinflammation.

Sexual Dimorphism in Extracellular Matrix Composition and Viscoelasticity of the Healthy and Inflamed Mouse Brain

“…Under neuroinflammatory conditions, we observed a mild effect in females and a significant softening of the midbrain of 5% in male EAE, although previous studies reported a global softening in female EAE brains [17][18][19] as well as for MS patients of both sexes [12]. This discrepancy could be explained by the limitation of the analyzed region, which was restricted to a single coronal slice, and did not cover the usually affected cerebellum or optic nerve [63,64].…”

“…To comply with animal welfare guidelines, all mice with a score higher than 3 were euthanized and removed from the study ( n female = 2, n male = 1). The EAE model using SJL mice has been shown to be more suitable for cerebral MRE studies than EAE in C57BL/6, since PLP-induced EAE in SJL consistently leads to the development of brain lesions and to elasticity alterations in brain structures [ 17 , 18 , 19 , 32 , 33 ].…”

“…MRE acquisition was performed on a preclinical 7 Tesla MRI scanner (BioSpec, Bruker, Ettlingen, Germany) operated with ParaVision 6.1 software (Bruker, Billerica, MA, USA) using a 20-mm diameter 1H-RF quadrature volume coil (RAPID Biomedical, Rimpar, Germany). The set up was similar to that previously described [ 19 , 29 ]. EAE mice were scanned based on their individual score at the peak of disease around day 10–12 after immunization.…”

“…In response to inflammation, however, the brains of female MS patients exhibited stronger reduction in viscoelasticity compared to age-matched healthy males [ 12 , 15 , 16 ]. Similarly, in the MS mouse model, experimental autoimmune encephalomyelitis (EAE), we previously demonstrated a reduction in brain stiffness in sick mice [ 17 , 18 , 19 ]. This softening behavior seemed to be associated with inflammatory processes such as immune cell infiltration, demyelination, and loss of the blood–brain barrier (BBB) integrity, as well as remodeling of the extracellular matrix (ECM) [ 18 , 19 , 20 , 21 ].…”

“…Similarly, in the MS mouse model, experimental autoimmune encephalomyelitis (EAE), we previously demonstrated a reduction in brain stiffness in sick mice [ 17 , 18 , 19 ]. This softening behavior seemed to be associated with inflammatory processes such as immune cell infiltration, demyelination, and loss of the blood–brain barrier (BBB) integrity, as well as remodeling of the extracellular matrix (ECM) [ 18 , 19 , 20 , 21 ]. We also recently showed that brain viscoelastic properties seem to be particularly dependent on ECM organization and axonal structure [ 22 ].…”