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. 2020 Jul 8;18(1):277.
doi: 10.1186/s12967-020-02441-6.

The brains of aged mice are characterized by altered tissue diffusion properties and cerebral microbleeds

Erik N Taylor  1   2   3 , Nasi Huang  4   5 , Jonathan Wisco  6 , Yandan Wang  7 , Kathleen G Morgan  7 , James A Hamilton  8   9
Affiliations

The brains of aged mice are characterized by altered tissue diffusion properties and cerebral microbleeds

Erik N Taylor et al. J Transl Med. .

Abstract

Background: Brain aging is a major risk factor in the progression of cognitive diseases including Alzheimer's disease (AD) and vascular dementia. We investigated a mouse model of brain aging up to 24 months old (mo).

Methods: A high field (11.7T) MRI protocol was developed to characterize specific features of brain aging including the presence of cerebral microbleeds (CMBs), morphology of grey and white matter, and tissue diffusion properties. Mice were selected from age categories of either young (3 mo), middle-aged (18 mo), or old (24 mo) and fed normal chow over the duration of the study. Mice were imaged in vivo with multimodal MRI, including conventional T2-weighted (T2W) and T2*-weighted (T2*W) imaging, followed by ex vivo diffusion-weighted imaging (DWI) and T2*W MR-microscopy to enhance the detection of microstructural features.

Results: Structural changes observed in the mouse brain with aging included reduced cortical grey matter volume and enlargement of the brain ventricles. A remarkable age-related change in the brains was the development of CMBs found starting at 18 mo and increasing in total volume at 24 mo, primarily in the thalamus. CMBs presence was confirmed with high resolution ex vivo MRI and histology. DWI detected further brain tissue changes in the aged mice including reduced fractional anisotropy, increased radial diffusion, increased mean diffusion, and changes in the white matter fibers visualized by color-coded tractography, including around a large cortical CMB.

Conclusions: The mouse is a valuable model of age-related vascular contributions to cognitive impairment and dementia (VCID). In composite, these methods and results reveal brain aging in older mice as a multifactorial process including CMBs and tissue diffusion alterations that can be well characterized by high field MRI.

Keywords: Aging; Brain imaging; Cerebral microbleeds (CMBs); Gradient-recalled echo MRI, diffusion tensor imaging; Vascular contributions to cognitive impairment and dementia (VCID).

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
Changes in brain structure occurring with aging in C57BL/6J mice. In vivo T2W MRI images are shown with single-slices from young (3 mo) or old (24 mo) mice in a and multi-slice maximum intensity projections (MaxIP) in d. The single-slice rostral view (a) is labeled with the cortex (Ct; green), hippocampus (Hi; blue), thalamus (red) and corpus callosum (Cc; purple) visible from a young and old mouse. b Quantification was carried out as linear measurements of Ct thickness superior to the Hi and Cc, across multiple locations and slices. Examples of Ct thickness measurements in the respective age groups are shown (top young mouse; bottom old mouse) with inset left hemisphere region, where the young mouse Ct thickness was about 1 mm. c The area of the two lateral ventricles was measured from the MaxIP images (*p value is less than 0.05 and #p value is less than 0.01 by ANOVA followed by Tukey multiple pairwise-comparisons). The white bar in d represents a scale bar with size of 5 mm
Fig. 2
Fig. 2
T2*W MRI located the hemosiderin deposits of CMBs in vivo. a A large CMB region was visible in the old-aged mouse is labeled with * in the MRI slice shown. b The relationship found between CMBs presence visible in T2*W MRI and the age of mice in months was significant in the linear regression model (p < 0.001; R2 value of 0.8716). The volume of CMBs was measured in vivo manually as the area of CMBs on an image multiplied by the slice thickness. Resolution (140 µm3 isotropic) was minimized to prevent partial volume effects. Quantification indicated increasing presence of CMBs in relation with aging (# indicates a p value is less than 0.01 by ANOVA followed by Tukey multiple pairwise-comparisons). The white bar represents a scale bar with size of 5 mm
Fig. 3
Fig. 3
Voxel-based morphometry (VBM) and regional brain analysis. VBM was performed using high resolution ex vivo T2*W MRI, demonstrating a statistical reduction in grey matter volume, particularly in the cerebral cortex. a The combined grey matter mask and Jacobian deformation field for an individual old mouse used for VBM analysis after linear and non-linear co-registration and spatial normalization in ANTs. Intensity is mapped to deformation, where red and yellow (positive) or green and blue (negative), respectively, progressively indicated morphologic changes in a representative old-aged mouse. White arrows are representative areas of cortical thinning. b A grey-scale template is shown, created from multiple co-registered, normalized, and intensity averaged young mouse brains. The areas where the voxels are significantly different in the young from the old mouse brains, as a combination of the Jacobian Field and grey-matter mask, are overlaid in red, with p < 0.05 (t-test). The white bar represents a scale bar with size of 2 mm
Fig. 4
Fig. 4
White matter (WM) fiber tractography and DTI changes ex vivo in young versus old C57BL/6J mouse brains. a WM fiber tractography in representative 3 mo (Young) and 24 mo (Old) male mice from high resolution ex vivo diffusion MRI scans with B = 2000 s/mm2, voxel size of 175 μm3, and 91 gradient directions. Whole brain DTI with tractography is shown where FA value is mapped to location. Tractography in the thalamus is displayed in the insets. b Measurements of the diffusion properties in the thalamus and parietal–temporal cortex ex vivo. DTI measurements taken from the respective ROI’s included FA, MD, and RD (in units of 10−3 mm2/s). The FA values in the thalamus/cortex decreased by 68%/67%, MD increased by 140%/134%, and RD increased by 151/139%, respectively, when comparing the 3 mo young mouse brains to old-aged mice (*p value is less than 0.05 when comparing the young mice to the old aged mice group). c Anomalous diffusion in diffusion MRI in relation to a CMB site observed with T2*W imaging. The CMB location is marked with an arrow. Radial diffusion (RD) was elevated in brain tissue surrounding the CMB. Local tractography was performed using three regions of interest (ROI) either on the same side (green), contralateral (orange), or through the site of the CMB (red). A region of elevated RD around the CMB region (indicated by the arrow) was found by mapping tractography to RD value (zero is blue and 0.5*10−3 mm2/s red, scale shown as inset). The white matter tracts passing through the CMBs site were lost. Whole brain tractography in relation to tractography through the ROI sites is shown in inset
Fig. 5
Fig. 5
CMBs shown with high resolution T2*W MR-microscopy from ex vivo brains (n = 16 total). a The majority of CMBs were found in the thalamus, the relay center of the brain, as shown in a representative minimum intensity projections (MinIP) from multiple slices of T2* MRI ex vivo. The C57BL/6J mice are shown in MinIP with the thalamus (Th), striatum (St), Hi visible from a young (3 mo) and old (24 mo) mouse scanned ex vivo after soaking in Gd contrast for 5 days. The arrow points to multiple thalamic CMBs across multiple slices. A single slice through the region indicated by the arrow is shown as an inset. b Multiple-planar views and high resolution isotropic images (55 µm3) enabled distinction and counting of individual CMBs. CMBs numbers increased in middle-aged and old mice with no significant differences between the number of CMBs in old-aged mice by sex. ANOVA testing indicated a p value less then 0.01 for CMBs by age, followed by the Tukey multiple pairwise-comparisons test, where #p value of less than 0.01 comparing young-aged to old and *p value of less then 0.05 when comparing middle-aged to old-aged mice for the CMBs count ex vivo
Fig. 6
Fig. 6
Histologic location of CMBs and analysis of regional blood supply. a Prussian blue histology of CMBs in an old mouse with the Th location of CMBs shown. The blood supply in microCT (blue) to the Th shown with MRI (grey) includes b the rostral Th, with supply from the internal carotid (IC) with the dorsal and ventral thalamic arteries. The MRI slice location is similar to the location of common Th CMBs, with the Th, St, and Hi labeled from MRI. c The blood supply caudal to the Th is shown with the posterior cerebral artery (PCA), connecting via the thalamoperforating arteries. The large central vessels visible superior to the Th in b, c are longitudinal hippocampal veins. Images in b, c were reproduced from data acquired by Dorr et al. [39]) using 7.0-T MRI with 32 μm isotropic voxels and microCT with 20 μm isotropic resolution in male CBA mice ex vivo with a scale bar size of 5 mm and the slice location shown as yellow bar on inset horizontal slice
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