Please wait a minute...
 Home  About the Journal Editorial Board Aims & Scope Peer Review Policy Subscription Contact us
Early Edition  //  Current Issue  //  Open Special Issues  //  Archives  //  Most Read  //  Most Downloaded  //  Most Cited
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
Download: PDF(1299 KB)   HTML
Export: BibTeX | EndNote | Reference Manager | ProCite | RefWorks    

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   
About author:

The authors contributed equally to this work.

Issue Date: 01 April 2018
E-mail this article
E-mail Alert
Articles by authors
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.
URL:     OR
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.
[1] Kalaria RN (2012). Cerebrovascular disease and mechanisms of cognitive impairment: evidence from clinicopathological studies in humans. Stroke, 43: 2526-2534
[2] Oakley R,Tharakan B (2014). Vascular hyperpermeability and aging. Aging Dis, 5: 114-125
[3] Gupta A,Nair S,Schweitzer AD,Kishore S,Johnson CE,Comunale JP,et al. (2012). Neuroimaging of cerebrovascular disease in the aging brain. Aging Dis, 3: 414-425
[4] Lin X,Miao P,Mu Z,Jiang Z,Lu Y,Guan Y,et al. (2015). Development of functional in vivo imaging of cerebral lenticulostriate artery using novel synchrotron radiation angiography. Phys Med Biol, 60: 1655-1665
[5] Iadecola C (2004). Neurovascular regulation in the normal brain and in Alzheimer’s disease. Nat Rev Neurosci, 5: 347-360
[6] Tang Y,Wang L,Wang J,Lin X,Wang Y,Jin K,et al. (2016). Ischemia-induced Angiogenesis is Attenuated in Aged Rats. Aging Dis, 7: 326-335
[7] Ertürk A,Mauch CP,Hellal F,Forstner F,Keck T,Becker K,et al. (2011). Three-dimensional imaging of the unsectioned adult spinal cord to assess axon regeneration and glial responses after injury. Nat Med, 18: 166-171
[8] Ragan T,Sylvan JD,Kim KH,Huang H,Bahlmann K,Lee RT,et al. (2007). High-resolution whole organ imaging using two-photon tissue cytometry. J Biomed Opt, 12: 014015
[9] Ragan T,Kadiri LR,Venkataraju KU,Bahlmann K,Sutin J,Taranda J,et al. (2012). Serial two-photon tomography for automated ex vivo mouse brain imaging. Nat Methods, 9: 255-258
[10] Mayerich D,Abbott L,McCormick B (2008). Knife-edge scanning microscopy for imaging and reconstruction of three-dimensional anatomical structures of the mouse brain. J Microsc, 231: 134-143
[11] Li A,Gong H,Zhang B,Wang Q,Yan C,Wu J,et al. (2010). Micro-optical sectioning tomography to obtain a high-resolution atlas of the mouse brain. Science, 330: 1404-1408
[12] Wu J,He Y,Yang Z,Guo C,Luo Q,Zhou W,et al. (2014). 3D BrainCV: simultaneous visualization and analysis of cells and capillaries in a whole mouse brain with one-micron voxel resolution. Neuroimage, 87: 199-208
[13] Wu J,Guo C,Chen S,Jiang T,He Y,Ding W,et al. (2016). Direct 3D Analyses Reveal Barrel-Specific Vascular Distribution and Cross-Barrel Branching in the Mouse Barrel Cortex. Cereb Cortex, 26: 23-31
[14] Osten P,Margrie TW (2013). Mapping brain circuitry with a light microscope. Nat Methods, 10: 515-523
[15] Hama H,Kurokawa H,Kawano H,Ando R,Shimogori T,Noda H,et al. (2011). Scale: a chemical approach for fluorescence imaging and reconstruction of transparent mouse brain. Nat Neurosci, 14: 1481-1488
[16] Kuwajima T,Sitko AA,Bhansali P,Jurgens C,Guido W,Mason C (2013). ClearT: a detergent- and solvent-free clearing method for neuronal and non-neuronal tissue. Development, 140: 1364-1368
[17] Ertürk A,Bradke F (2013). High-resolution imaging of entire organs by 3-dimensional imaging of solvent cleared organs (3DISCO). Exp Neurol, 242: 57-64
[18] Ertürk A,Becker K,Jahrling N,Mauch CP,Hojer CD,Egen JG,et al. (2012). Three-dimensional imaging of solvent-cleared organs using 3DISCO. Nat Protoc, 7: 1983-1995
[19] Ke MT,Fujimoto S,Imai T (2013). SeeDB: a simple and morphology-preserving optical clearing agent for neuronal circuit reconstruction. Nat Neurosci, 16: 1154-1161
[20] Ke MT,Imai T (2014). Optical clearing of fixed brain samples using SeeDB. Curr Protoc Neurosci, 66: Unit 2 22
[21] Fumoto S,Nishimura K,Nishida K,Kawakami S (2016). Three-Dimensional Imaging of the Intracellular Fate of Plasmid DNA and Transgene Expression: ZsGreen1 and Tissue Clearing Method CUBIC Are an Optimal Combination for Multicolor Deep Imaging in Murine Tissues. PloS One, 11: e0148233
[22] Susaki EA,Tainaka K,Perrin D,Kishino F,Tawara T,Watanabe TM,et al. (2014). Whole-brain imaging with single-cell resolution using chemical cocktails and computational analysis. Cell, 157: 726-739
[23] Susaki EA,Tainaka K,Perrin D,Yukinaga H,Kuno A,Ueda HR (2015). Advanced CUBIC protocols for whole-brain and whole-body clearing and imaging. Nat Protoc, 10: 1709-1727
[24] Renier N,Wu Z,Simon DJ,Yang J,Ariel P,Tessier-Lavigne M (2014). iDISCO: a simple, rapid method to immunolabel large tissue samples for volume imaging. Cell, 159: 896-910
[25] Chung K,Deisseroth K (2013). CLARITY for mapping the nervous system. Nat Methods, 10: 508-513
[26] Tomer R,Ye L,Hsueh B,Deisseroth K (2014). Advanced CLARITY for rapid and high-resolution imaging of intact tissues. Nat Protoc, 9: 1682-1697
[27] Chung K,Wallace J,Kim SY,Kalyanasundaram S,Andalman AS,Davidson TJ,et al. (2013). Structural and molecular interrogation of intact biological systems. Nature, 497: 332-337
[28] Spence RD,Kurth F,Itoh N,Mongerson CR,Wailes SH,Peng MS,et al. (2014). Bringing CLARITY to gray matter atrophy. Neuroimage, 101: 625-632
[29] Huang J,Li Y,Tang Y,Tang G,Yang GY,Wang Y (2013). CXCR4 antagonist AMD3100 protects blood-brain barrier integrity and reduces inflammatory response after focal ischemia in mice. Stroke, 44: 190-197
[30] Yang B,Treweek JB,Kulkarni RP,Deverman BE,Chen CK,Lubeck E,et al. (2014). Single-cell phenotyping within transparent intact tissue through whole-body clearing. Cell, 158: 945-958
[31] Peng H,Ruan Z,Long F,Simpson JH,Myers EW (2010). V3D enables real-time 3D visualization and quantitative analysis of large-scale biological image data sets. Nat Biotechnol, 28: 348-353
[32] Xiao H,Peng H (2013). APP2: automatic tracing of 3D neuron morphology based on hierarchical pruning of a gray-weighted image distance-tree. Bioinformatics, 29: 1448-1454
[33] Peng H,Ruan Z,Atasoy D,Sternson S (2010). Automatic reconstruction of 3D neuron structures using a graph-augmented deformable model. Bioinformatics, 26: i38-46
[34] Tsai PS,Kaufhold JP,Blinder P,Friedman B,Drew PJ,Karten HJ,et al. (2009). Correlations of neuronal and microvascular densities in murine cortex revealed by direct counting and colocalization of nuclei and vessels. J Neurosci, 29: 14553-14570
[35] Ren C,Li N,Wang B,Yang Y,Gao J,Li S,et al. (2015). Limb Ischemic Perconditioning Attenuates Blood-Brain Barrier Disruption by Inhibiting Activity of MMP-9 and Occludin Degradation after Focal Cerebral Ischemia. Aging Dis, 6: 406-417
[36] Marx V (2014). Microscopy: seeing through tissue. Nat Methods, 11: 1209-1214
[37] Feng Y,Cui P,Lu X,Hsueh B,Moller Billig F,Zarnescu Yanez L,et al. (2017). CLARITY reveals dynamics of ovarian follicular architecture and vasculature in three-dimensions. Sci Rep, 7: 44810
[38] Short KM,Combes AN,Lefevre J,Ju AL,Georgas KM,Lamberton T,et al. (2014). Global quantification of tissue dynamics in the developing mouse kidney. Dev Cell, 29: 188-202
[39] Ali RA,Landsberg MJ,Knauth E,Morgan GP,Marsh BJ,Hankamer B (2012). A 3D image filter for parameter-free segmentation of macromolecular structures from electron tomograms. PloS One, 7: e33697
[40] Yang X,Liu C,Le Minh H,Wang Z,Chien A,Cheng KT (2017). An automated method for accurate vessel segmentation. Phys Med Biol, 62: 3757-3778
[41] Chen C,Lin X,Wang J,Tang G,Mu Z,Chen X,et al. (2014). Effect of HMGB1 on the paracrine action of EPC promotes post-ischemic neovascularization in mice. Stem Cells, 32: 2679-2689
[42] Ergul A,Alhusban A,Fagan SC (2012). Angiogenesis: a harmonized target for recovery after stroke. Stroke, 43: 2270-2274
[43] Busch HJ,Buschmann IR,Mies G,Bode C,Hossmann KA (2003). Arteriogenesis in hypoperfused rat brain. J Cereb Blood Flow Metab, 23: 621-628
[44] Buschmann I,Heil M,Jost M,Schaper W (2003). Influence of inflammatory cytokines on arteriogenesis. Microcirculation, 10: 371-379
[45] Tanaka Y,Nagaoka T,Nair G,Ohno K,Duong TQ (2011). Arterial spin labeling and dynamic susceptibility contrast CBF MRI in postischemic hyperperfusion, hypercapnia, and after mannitol injection. J Cereb Blood Flow Metab, 31: 1403-1411
[46] Boero JA,Ascher J,Arregui A,Rovainen C,Woolsey TA (1999). Increased brain capillaries in chronic hypoxia. J Appl Physiol, 86: 1211-1219
[47] Heinzer S,Krucker T,Stampanoni M,Abela R,Meyer EP,Schuler A,et al. (2006). Hierarchical microimaging for multiscale analysis of large vascular networks. Neuroimage, 32: 626-636
[48] Heinzer S,Kuhn G,Krucker T,Meyer E,Ulmann-Schuler A,Stampanoni M,et al. (2008). Novel three-dimensional analysis tool for vascular trees indicates complete micro-networks, not single capillaries, as the angiogenic endpoint in mice overexpressing human VEGF(165) in the brain. Neuroimage, 39: 1549-1558
[49] Serduc R,Verant P,Vial JC,Farion R,Rocas L,Remy C,et al. (2006). In vivo two-photon microscopy study of short-term effects of microbeam irradiation on normal mouse brain microvasculature. Int J Radiat Oncol Biol Phys, 64: 1519-1527
[50] Verant P,Serduc R,Van Der Sanden B,Remy C,Vial JC (2007). A direct method for measuring mouse capillary cortical blood volume using multiphoton laser scanning microscopy. J Cereb Blood Flow Metab, 27: 1072-1081
[51] Zhao R,Pollack GM (2009). Regional differences in capillary density, perfusion rate, and P-glycoprotein activity: a quantitative analysis of regional drug exposure in the brain. Biochem Pharmacol, 78: 1052-1059
[52] Jolivel V,Bicker F,Biname F,Ploen R,Keller S,Gollan R,et al. (2015). Perivascular microglia promote blood vessel disintegration in the ischemic penumbra. Acta Neuropathol, 129: 279-295
[1] Yali Chen, Mengmei Yin, Xuejin Cao, Gang Hu, Ming Xiao. Pro- and Anti-inflammatory Effects of High Cholesterol Diet on Aged Brain[J]. Aging and disease, 2018, 9(3): 374-390.
[2] Can Zhang,Nicole R. Brandon,Kerryann Koper,Pei Tang,Yan Xu,Huanyu Dou. Invasion of Peripheral Immune Cells into Brain Parenchyma after Cardiac Arrest and Resuscitation[J]. A&D, 2018, 9(3): 412-425.
[3] Peng Fangyu, Xie Fang, Muzik Otto. Alteration of Copper Fluxes in Brain Aging: A Longitudinal Study in Rodent Using 64CuCl2-PET/CT[J]. Aging and disease, 2018, 9(1): 109-118.
[4] Liu Huaqin, Yu Zhui, Li Ying, Xu Bin, Yan Baihui, Paschen Wulf, Warner David S, Yang Wei, Sheng Huaxin. Novel Modification of Potassium Chloride Induced Cardiac Arrest Model for Aged Mice[J]. Aging and disease, 2018, 9(1): 31-39.
[5] Farnaz Farokhian,Chunlan Yang,Iman Beheshti,Hiroshi Matsuda,Shuicai Wu. Age-Related Gray and White Matter Changes in Normal Adult Brains[J]. A&D, 2017, 8(6): 899-909.
[6] Ulises Urzua,Carlos Chacon,Luis Lizama,Sebastián Sarmiento,Pía Villalobos,Belén Kroxato,Katherine Marcelain,María-Julieta Gonzalez. Parity History Determines a Systemic Inflammatory Response to Spread of Ovarian Cancer in Naturally Aged Mice[J]. A&D, 2017, 8(5): 546-557.
[7] Rongliang Wang,Jincheng Li,Yunxia Duan,Zhen Tao,Haiping Zhao,Yumin Luo. Effects of Erythropoietin on Gliogenesis during Cerebral Ischemic/Reperfusion Recovery in Adult Mice[J]. A&D, 2017, 8(4): 410-419.
[8] Giuseppe Pasqualetti,Marta Seghieri,Eleonora Santini,Chiara Rossi,Edoardo Vitolo,Livia Giannini,Maria Giovanna Malatesta,Valeria Calsolaro,Fabio Monzani,Anna Solini. P2X7 Receptor and APOE Polymorphisms and Survival from Heart Failure: A Prospective Study in Frail Patients in a Geriatric Unit[J]. A&D, 2017, 8(4): 434-441.
[9] Diana L Castillo-Carranza,Ashley N Nilson,Candice E Van Skike,Jordan B Jahrling,Kishan Patel,Prajesh Garach,Julia E Gerson,Urmi Sengupta,Jose Abisambra,Peter Nelson,Juan Troncoso,Zoltan Ungvari,Veronica Galvan,Rakez Kayed. Cerebral Microvascular Accumulation of Tau Oligomers in Alzheimer’s Disease and Related Tauopathies[J]. A&D, 2017, 8(3): 257-266.
[10] Jianhui Wang,Xiaorui Cheng,Ju Zeng,Jiangbei Yuan,Zhongfu Wang,Wenxia Zhou,Yongxiang Zhang. LW-AFC Effects on N-glycan Profile in Senescence-Accelerated Mouse Prone 8 Strain, a Mouse Model of Alzheimer’s Disease[J]. A&D, 2017, 8(1): 101-114.
[11] Yunpeng Lin,Lan Lan Luo,Jian Sun,Weiwei Gao,Ye Tian,Eugene Park,Andrew Baker,Jieli Chen,Rongcai Jiang,Jianning Zhang. Relationship of Circulating CXCR4+ EPC with Prognosis of Mild Traumatic Brain Injury Patients[J]. A&D, 2017, 8(1): 115-127.
[12] Deyong Lv,Jingbo Li,Hongfu Li,Yu Fu,Wei Wang. Imaging and Quantitative Analysis of the Interstitial Space in the Caudate Nucleus in a Rotenone-Induced Rat Model of Parkinson’s Disease Using Tracer-based MRI[J]. A&D, 2017, 8(1): 1-6.
[13] Mei-Yan Liu,Yan-Ping Ren,Li-Jun Zhang,Jamie Y. Ding. Pretreatment with Ginseng Fruit Saponins Affects Serotonin Expression in an Experimental Comorbidity Model of Myocardial Infarction and Depression[J]. A&D, 2016, 7(6): 680-686.
[14] Lei Su,Yujuan Han,Rong Xue,Kristofer Wood,Fu-Dong Shi,Yaou Liu,Ying Fu. Thalamic Atrophy Contributes to Low Slow Wave Sleep in Neuromyelitis Optica Spectrum Disorder[J]. A&D, 2016, 7(6): 691-696.
[15] Yaohui Tang,Liuqing Wang,Jixian Wang,Xiaojie Lin,Yongting Wang,Kunlin Jin,Guo-Yuan Yang. Ischemia-induced Angiogenesis is Attenuated in Aged Rats[J]. A&D, 2016, 7(4): 326-335.
Full text



Copyright © 2014 Aging and Disease, All Rights Reserved.
Address: Aging and Disease Editorial Office 3400 Camp Bowie Boulevard Fort Worth, TX76106 USA
Fax: (817) 735-0408 E-mail:
Powered by Beijing Magtech Co. Ltd