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Molecular profiles underlying the fluid-solid transition of brain tissue during maturation: High-resolution multifrequency MR elastography of the mouse brain paired with proteomics mass spectrometry.1Department of Radiology, Charité - Universitätsmedizin Berlin, Berlin, Germany, 2Department of Neurology, Charité - Universitätsmedizin Berlin, Berlin, Germany, 3Max Planck Institute for Molecular Genetics, Berlin, Germany, 4Department for Medical Immunology, Charité - Universitätsmedizin Berlin, Berlin, Germany, 5Department of Medical Informaticsy, Charité - Universitätsmedizin Berlin, Berlin, Germany
Synopsis
We have investigated the mechanical development of the mouse brain from 4 weeks to 20 weeks by multifrequency MR elastography and performed proteomics analysis with mass spectrometry. Highly resolved elastographic atlases of brain maturation were generated and regional analysis was performed to identify areas with age-dependent changes in viscoelasticity. In hippocampus, molecular signatures associated with macroscopically observed mechanical profile were identified, suggesting a transition from a soft-fluid to a more elastic-solid state during maturation. Combining MRE and proteomic analysis, we provide structural and functional information related to brain maturation from the molecular level up to whole-organ mechanical scale.
Introduction
Magnetic resonance elastography (MRE)1 measures in-vivo the mechanical properties of the mouse brain2. Recent findings demonstrated that tissue mechanical properties reveal information about tissue structures that change with aging and disease3, 4. However, little is known about the microstructural origin of mechanical parameter changes during brain maturation. In this study, we aimed at generating i) age-dependent elastographic mouse brain atlas based on tomoelastography5, ii) protein profiles of brain tissue by mass spectrometry (MS), and iii) correlations of MRE with MS and histological analysis to identify structural proteins that affect the macroscopic viscoelastic properties.Methods
Experiments were performed on 32 female wild-type C57BL/6 mice at 4,8,12,16 and 20 weeks of age. At each time point, 5 mice were sacrificed after MRE for proteomic and histological analysis. MRE was performed on a 7-T small-animal scanner (Bruker, Biospec, Ettlingen, Germany) using 5 frequencies (1000,1100,1200,1300 and 1400 Hz). The vibration was generated by a piezoceramic actuator and transferred to the head via a head holder5. 3D-wave fields were acquired by single-shot spin-echo-EPI5. Acquisition time for 9 slices of 0.2×0.2×1mm3 resolution, 5 frequencies, 8 wave dynamics, 3 signal averagings was 4.5 minutes. MRE data were processed by k-MDEV-inversion6, yielding parameter maps of shear wave speed (c) surrogating tissue stiffness. Additionally, maps of phase angle (φ) of the complex shear modulus, which is related to the viscoelastic dispersion of the tissue, were computed by MDEV-inversion7. Individual c and φ-maps were normalized to the Allen mouse brain atlas using anatomical T2w images. Thirteen anatomical atlas regions were analyzed. Proteomics analysis was performed using the right hippocampal tissue while the left hemisphere was used for myelin quantification by Luxol fast blue staining and neurogenesis quantification by doublecortin (DCX) staining.Results
Multifrequency MRE with tomoelastography data processing provided highly resolved maps of stiffness and viscosity (c-maps and φ-maps) of the mouse brain (Fig.1). Fig.1a depicts normalized parameter maps averaged over 10 mice at five ages and shows that hippocampus and midbrain stiffen during maturation. Fig.1b shows fine anatomical details such as layers of somatosensory cortex and motor cortex. Further subregional analysis revealed that brain stiffness (c) was significantly age-dependent in twelve brain regions named in the caption of Fig.2, which presents variations of MRE parameters and protein levels as log2-fold changes relative to 4 weeks. All twelve areas in Fig.2a stiffened over age except of cortical regions. Fig.2b lists six regions where viscosity (φ) was significantly altered by age. A monotonic reduction was only observed in hippocampus and corpus callosum. In hippocampus, stiffness and viscosity at all ages correlated with 13 and 9 age-regulated proteins in function groups of structural plasticity and cell adhesion7, respectively (Fig.3). Hippocampal myelin content estimated from histological staining was well correlated with Myelin-oligodendrocyte glycoprotein (MOG) level obtained by MS (Pearson r=0.57). A reduced neurogenesis with age was indicated by the decrease in DCX+ cells. Microscope images of myelin and DCX staining are shown in Fig.4.Discussion
To our knowledge, this is the first presentation of age-dependent high-resolution tomoelastography atlases of the mouse brain. In-vivo viscoelasticity data were paired with proteomics to identify microstructural components which are relevant for the macroscopic cerebral viscoelasticity. During maturation, stiffness of the whole brain increased slightly by 3% while viscosity (φ) remained unchanged, which is consistent with8. However, distinct age-regulation were found in different brain regions. In hippocampus, variations in structural proteins were directly linked to the observed changes in mechanical properties. We found that upregulation of proteins related to microtubular structures, myelination, cytoskeleton linkage and cell-ECM attachment (e.g. Nefh, Mapt, Plec, Mog) are responsible for hippocampal stiffening. Positive correlation between myelination and brain stiffness was consistence with observations made in9. Reduced viscosity was correlated with diminished actin crosslink and axonal organization as reflected by the down-regulation of structural proteins such as DPYSL, Robo2 and Tnc. Altogether, we have identified multifactorial influences on the observed mechanical properties and our data suggested an in-vivo transition of hippocampal tissue from a more soft-fluid towards more elastic-solid state in the course of maturation driven by myelination and cellular organization.Conclusion
We presented highly resolved age-dependent atlases of viscoelastic parameters of the mouse brain and have identified molecular signatures associated with increased macroscopic stiffness and reduced viscosity which indicated hippocampal transition from a soft-fluid to a more elastic-solid state during maturation. These cerebral mechanical profiles might direct future MRE application to diseases which involve molecules our study has identified as relevant for brain mechanics to make the diagnosis of cerebral MRE more specific.Acknowledgements
The work was funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) – GRK2260 (BIOQIC) and SFB 1340/1 2018.References
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