Demyelination reduces brain parenchymal stiffness quantified in vivo by magnetic resonance elastography

Abstract

The detection of pathological tissue alterations by manual palpation is a simple but essential diagnostic tool, which has been applied by physicians since the beginnings of medicine. Recently, the virtual "palpation" of the brain has become feasible using magnetic resonance elastography, which quantifies biomechanical properties of the brain parenchyma by analyzing the propagation of externally elicited shear waves. However, the precise molecular and cellular patterns underlying changes of viscoelasticity measured by magnetic resonance elastography have not been investigated up to date. We assessed changes of viscoelasticity in a murine model of multiple sclerosis, inducing reversible demyelination by feeding the copper chelator cuprizone, and correlated our results with detailed histological analyses, comprising myelination, extracellular matrix alterations, immune cell infiltration and axonal damage. We show firstly that the magnitude of the complex shear modulus decreases with progressive demyelination and global extracellular matrix degradation, secondly that the loss modulus decreases faster than the dynamic modulus during the destruction of the corpus callosum, and finally that those processes are reversible after remyelination.

Fig. 1.

T2-weighted MRI (above) and reconstructed maps of the complex-valued shear modulus |G*| (below). (Left) A healthy control. (Right) A mouse after 12 wk of continuous cuprizone diet. During treatment, the T2 signal intensity of the myelinated corpus callosum (arrowheads) became isointense to the neighboring gray matter, and viscoelasticity showed a significant decrease in the region-of-interest (marked by the red line), corresponding to progressive demyelination.

Fig. 2.

Quantitative analysis of T2-weighted signal intensity (A), phase-angle y (B) and viscoelasticity |G*| (C) in the corpus callosum. Longitudinal data in healthy control mice (HC; white bars), cuprizone-fed mice (CUP; black bars), and mice discontinuing cuprizone diet after 9 wk (gray bars) are presented. (A) T2-weighted signal intensity was computed as ROI ratio of corpus callosum and thalami. This ratio developed reciprocally in HC and CUP: Although, initially, the two groups did not differ (week 0), T2-signal intensity decreased significantly in HC (P < 0.0001), but increased progressively in CUP, reaching a maximum at week 12. (B) Whereas y was constant in HC, it decreased progressively in CUP, reaching a minimum in week 12. (C) Viscoelasticity rose in HC and CUP between weeks 3 and 9; however, this increase was less pronounced in CUP. |G*| reached a plateau in HC, but decreased significantly in CUP at week 12. Mice returning to normal diet at week 9 partially recuperated. **P < 0.001, ***P < 0.0001; treated mice compared with HC in Bonferroni-corrected one- or two-way ANOVA at every time point (in consequence, α was lowered and results were classified as significant when P value was < 0.025). SD by whiskers.

Fig. 3.

To assess demyelination, brain sections were immunohistochemically stained for myelin basic protein (MBP). (A) Overview and magnification of the corpus callosum (arrows) in a healthy control (HC; left side) and a mouse after 12 wk on cuprizone diet (CUP; right side). HC stained homogeneously with intact myelin sheaths. In contrast, CUP at week 12 showed almost complete demyelination. (B) Semiquantitative analysis of demyelination with scores ranging from 4 = normal myelination, to 0 = no myelin. Pooled data of HC are given (white bar). In CUP, demyelination progressed constantly and was almost complete after 12 wk of cuprizone feeding (black bars). The subgroup of mice returning to normal chow after 9 wk of cuprizone diet showed incomplete remyelination at week 12 (gray bar). ***P < 0.0001; CUP compared with HC in an unpaired t test at every time point (in consequence, α was lowered and results were classified as significant when P value was < 0.01). SD by whiskers.

Fig. 4.

(A) Extracellular matrix (ECM) alterations were studied by hematoxylin and eosin (H&E, Top), Alcian blue (AB, Middle), and immunohistochemical fibronectin (FN) stainings (Bottom). The corpus callosum of healthy controls (HC; Left) was homogenous and showed an intact tissue structure on all stainings. AB (glycosaminoglycans, mucopolysaccharides) and FN were barely present in the corpus callosum of HC. In contrast, the parenchyma of mice after 12 wk on cuprizone diet (CUP; Right) was markedly altered. H&E depicted a heterogeneously composed ECM with vacuoles (arrows; Top Right) and eosinophilia. AB showed a patchy accumulations of glycosaminoglycans and mucopolysaccharides (blue staining, arrows; Middle Right). FN staining in the corpus callosum increased largely (brown staining; Bottom Right). An increasing cell density was evident in all stainings after cuprizone feeding. (B) Tissue integrity alterations were assessed applying a semiquantitative score in H&E, AB and FN staining. The score ranged from 0 (normal) to 3 (heavily altered ECM; vacuoles, disruption). Controls (white bar) were pooled. In CUP (black bars), alterations were constantly progressive and reached a maximum at week 12. In mice discontinuing the cuprizone diet after 9 wk (gray bar), ECM recuperated incompletely. The extent of AB (C) and FN (D) staining in the corpus callosum was evaluated with a score ranging from 0 (no staining) to 3 (intense staining). Controls (white bar) were pooled. In CUP (black bars), stainings were most pronounced after 12 wk of cuprizone diet. **P < 0.001, ***P < 0.0001; CUP compared with HC with an one-way ANOVA. SD by whiskers.

Fig. 5.

Astrogliosis was assessed by immunohistochemical staining for glial fibrillary acidic protein (GFAP). (A) Extensive astrogliosis was observed after 9 wk of cuprizone feeding (Right; Upper: green = GFAP, astrocytes; blue = Hoechst 33258, cell nuclei; red = fluoromyelin; Lower: gliosis, brown in DAB staining) in comparison with control mice (HC; Left). (B) Semiquantitative analysis of gliosis in the corpus callosum. The score ranges from 0 (none) to 4 (massive gliosis); HC were pooled (white bar). In treated mice (black bars), astrogliosis increased progressively until reaching a maximum at week 9, and declined thereafter. **P < 0.001, ***P < 0.0001; treated mice compared with HC in an unpaired t test at every time point (in consequence, α was lowered and results were classified as significant when P value was < 0.01). SD by whiskers.

Fig. 6.

Cuprizone feeding did not induce permanent neuronal damage. (A) The expression of β-amyloid precursor protein (β-APP) was transiently increased after 9 wk of cuprizone feeding compared with control mice (A Upper: red = β-APP; blue = Hoechst dye 33258, cell nuclei). Staining for neurofilament M was not altered in cuprizone fed mice. Accordingly, there was no evidence of relevant neuronal damage (A Lower: green = anti-phosphorylated neurofilament M antibody; blue = Hoechst dye 33258, cell nuclei). (B) Time course of β-APP expression (ovoids per mm²) expressed in control mice (white bar) compared with cuprizone-fed mice (black bars).