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Aging and disease    2018, Vol. 9 Issue (2) : 262-272     DOI: 10.14336/AD.2017.0613
Orginal Article |
CLARITY for High-resolution Imaging and Quantification of Vasculature in the Whole Mouse Brain
Zhang Lin-Yuan1, Lin Pan2, Pan Jiaji3, Ma Yuanyuan1, Wei Zhenyu4, Jiang Lu3, Wang Liping1, Song Yaying1, Wang Yongting3, Zhang Zhijun3, Jin Kunlin5, Wang Qian2,*, Yang Guo-Yuan1,3,*
1Department of Neurology, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China
2Medical Image Computing Lab and
3Neuroscience and Neuroengineering Research Center, Med-X Research Institute and School of Biomedical Engineering, Shanghai Jiao Tong University, Shanghai 200030, China
4Shanghai Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 201999, China
5Department of Pharmacology and Neuroscience, University of North Texas Health Science Center, TX76107, USA
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Abstract  

Elucidating the normal structure and distribution of cerebral vascular system is fundamental for understanding its function. However, studies on visualization and whole-brain quantification of vasculature with cellular resolution are limited. Here, we explored the structure of vasculature at the whole-brain level using the newly developed CLARITY technique. Adult male C57BL/6J mice undergoing transient middle cerebral artery occlusion and Tie2-RFP transgenic mice were used. Whole mouse brains were extracted for CLARITY processing. Immunostaining was performed to label vessels. Customized MATLAB code was used for image processing and quantification. Three-dimensional images were visualized using the Vaa3D software. Our results showed that whole mouse brain became transparent using the CLARITY method. Three-dimensional imaging and visualization of vasculature were achieved at the whole-brain level with a 1-μm voxel resolution. The quantitative results showed that the fractional vascular volume was 0.018 ± 0.004 mm3 per mm3, the normalized vascular length was 0.44 ± 0.04 m per mm3, and the mean diameter of the microvessels was 4.25 ± 0.08 μm. Furthermore, a decrease in the fractional vascular volume and a decrease in the normalized vascular length were found in the penumbra of ischemic mice compared to controls (p < 0.05). In conclusion, CLARITY provides a novel approach for mapping vasculature in the whole mouse brain at cellular resolution. CLARITY-optimized algorithms facilitate the assessment of structural change in vasculature after brain injury.

Keywords brain      clarity      imaging process      mouse      vasculature     
Corresponding Authors: Wang Qian,Yang Guo-Yuan   
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The authors contributed equally to this work.

Issue Date: 01 April 2018
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Zhang Lin-Yuan
Lin Pan
Pan Jiaji
Ma Yuanyuan
Wei Zhenyu
Jiang Lu
Wang Liping
Song Yaying
Wang Yongting
Zhang Zhijun
Jin Kunlin
Wang Qian
Yang Guo-Yuan
Cite this article:   
Zhang Lin-Yuan,Lin Pan,Pan Jiaji, et al. CLARITY for High-resolution Imaging and Quantification of Vasculature in the Whole Mouse Brain[J]. Aging and disease, 2018, 9(2): 262-272.
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http://www.aginganddisease.org/EN/10.14336/AD.2017.0613     OR     http://www.aginganddisease.org/EN/Y2018/V9/I2/262
Figure 1.  Processing procedures of clarified sample image. A) Original image obtained from the clarified sample. Scale bar=100 μm. B) Image preprocessing to uniform background intensity of the image. C) Vessel recognition by 3D Canny edge detection and morphology operation. D) Image segmentation and binarization. E) Vessel tracing using Vaa3D software. F) Visualization of 3D rendering images was performed by Vaa3D.
Figure 2.  CLARITY renders whole adult mouse brain transparent. A) Whole adult mouse brain before CLARITY process. B) Whole adult mouse brain after removing lipid bilayers. C) Whole adult mouse brain after refractive index matching. Scale bar=1 mm.
Figure 3.  Imaging of vasculature in the whole mouse brain after CLARITY. A) Whole brain vessels of Tie2-RFP transgenic mouse were visualized using a confocal microscopy. Scale bar=1 mm. B) Magnification of white box region in (A). Scale bar=500 μm. C) 3D reconstruction of the vasculature in a whole, clarified mouse brain. Images were obtained using confocal microscopy equipped with a 40× water-immersion objective. The imaging volume was 1120 μm×550 μm×3270 μm with a voxel size of 0.62 μm×0.62 μm×1.38 μm. (D-F) Images at different brain depths (1035 μm, 2070 μm, and 2760 μm relative to the top imaging surface) using a 40× water-immersion objective with a voxel size of 0.62 μm × 0.62 μm × 1.38 μm. Scale bar=100 μm.
Figure 4.  Imaging of vasculature in a 1-mm-thick mouse brain slice stained with lectin and claudin-5. A) 3D rendering of a 1-mm-thick wild-type C57BL/6J mouse brain slice stained with lectin-FITC. Images were obtained from confocal microscopy equipped with a 16× objective. The imaging volume was 4300 μm×5260 μm×880 μm, with a voxel size of 0.99 μm×0.99 μm×1.00 μm. Scale bar=1 mm. B) Magnification of the white box region in (A). The imaging volume was 1367 μm×682 μm×678 μm. C-E) Images at different depths of the brain slice from (B) are shown (125 μm, 340 μm, and 580 μm relative to the top imaging surface) using a 16× water-immersion objective with a voxel size of 0.99 μm×0.99 μm×1.00 μm. Scale bars=50 μm. (F) 3D rendering of a 1-mm-thick C57BL/6J mouse brain slice stained with claudin-5 antibody. Images were obtained using confocal microscopy equipped with a 16× objective. The imaging volume was 504 μm×504 μm×900 μm, with a voxel size of 0.99 μm×0.99 μm×1.00 μm. G) Magnification of white box region in (F). The imaging volume was 195 μm×195 μm×100 μm, with a voxel size of 0.99 μm×0.99 μm×1.00 μm.
Figure 5.  Visualization of vasculature in whole mouse brain after CLARITY. Visualization of 3D rendered images was performed using Vaa3D software and ewith 60°, 120°, 180°, 240°, 320°, and 360° anticlockwise rotations around the z-axis. Images were obtained using confocal microscopy equipped with a 40× water-immersion objective. The imaging volume was 1120 μm×550 μm×3270 μm, with a voxel size of 0.62 μm×0.62 μm×1.38 μm. A) 0°, B) 60° rotation, C) 120° rotation, D) 180° rotation, E) 240° rotation, F) 300° rotation, and G) 360° rotation.
Figure 6.  Visualization and quantification of the vasculature in the penumbra of tMCAO and sham mouse brain. A) Representative 3D image of the vasculature stained with claudin-5 in the penumbra of C57BL/6J mouse brain from the control and tMCAO group after 24 hours of reperfusion. The imaging volume was 504 μm×504 μm×886 μm, with a voxel size of 0.99 μm×0.99 μm×2.00 μm. Scale bar=250 μm. B) Mouse brain coronal section indicating core and penumbra region of the infarct area and the box indicating the area that was sampled. Quantification of the fractional vascular volume and the normalized vessel length in the controls and in the tMCAO group after 24 hours of reperfusion (C and D). Data are mean ± standard error, n=3 per group. *p < 0.05, tMCAO vs. control. tMCAO: transient middle cerebral artery occlusion.
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