Skip to main content

Advertisement

SpringerLink
Log in

A Novel Technique for Visualizing and Analyzing the Cerebral Vasculature in Rodents

  • Methods Paper
  • Published:
Translational Stroke Research Aims and scope Submit manuscript

Abstract

We introduce a novel protocol to stain, visualize, and analyze blood vessels from the rat and mouse cerebrum. This technique utilizes the fluorescent dye, DiI, to label the lumen of the vasculature followed by perfusion fixation. Following brain extraction, the labeled vasculature is then imaged using wide-field fluorescence microscopy for axial and coronal images and can be followed by regional confocal microscopy. Axial and coronal images can be analyzed using classical angiographic methods for vessel density, length, and other features. We also have developed a novel fractal analysis to assess vascular complexity. Our protocol has been optimized for adult rat, adult mouse, and neonatal mouse studies. The protocol is efficient, can be rapidly completed, stains cerebral vessels with a bright fluorescence, and provides valuable quantitative data. This method has a broad range of applications, and we demonstrate its use to study the vasculature in assorted models of acquired brain injury.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

References

  1. Cipolla MJ. The cerebral circulation. integrated systems physiology: from molecule to function. San Rafael (CA) 2009.

  2. Xing C, Arai K, Lo EH, Hommel M. Pathophysiologic cascades in ischemic stroke. Int J Stroke. 2012;7(5):378–85. https://doi.org/10.1111/j.1747-4949.2012.00839.x.

    Article  PubMed  PubMed Central  Google Scholar 

  3. Taoufik E, Probert L. Ischemic neuronal damage. Curr Pharm Des. 2008;14(33):3565–73.

    Article  CAS  PubMed  Google Scholar 

  4. Muoio V, Persson PB, Sendeski MM. The neurovascular unit—concept review. Acta Physiol (Oxf). 2014;210(4):790–8. https://doi.org/10.1111/apha.12250.

    Article  CAS  Google Scholar 

  5. Cauli B, Hamel E. Revisiting the role of neurons in neurovascular coupling. Front Neuroenerg. 2010;2:9. https://doi.org/10.3389/fnene.2010.00009.

    Article  Google Scholar 

  6. Hughes S, Dashkin O, Defazio RA. Vessel painting technique for visualizing the cerebral vascular architecture of the mouse. Methods Mol Biol. 2014;1135:127–38. https://doi.org/10.1007/978-1-4939-0320-7_12.

    Article  PubMed  Google Scholar 

  7. Li Y, Song Y, Zhao L, Gaidosh G, Laties AM, Wen R. Direct labeling and visualization of blood vessels with lipophilic carbocyanine dye DiI. Nat Protoc. 2008;3(11):1703–8. https://doi.org/10.1038/nprot.2008.172.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Moy AJ, Wiersma MP, Choi B. Optical histology: a method to visualize microvasculature in thick tissue sections of mouse brain. PLoS One. 2013;8(1):e53753. https://doi.org/10.1371/journal.pone.0053753.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Salehi A, Jullienne A, Baghchechi M, Hamer M, Walsworth M, Donovan V et al. Up-regulation of Wnt/β-catenin expression is accompanied with vascular repair after traumatic brain injury. J Cereb Blood Flow Metab 2018;38(2):274–89. https://doi.org/10.1177/0271678X17744124.

  10. Joshi VS, Reinhardt JM, Garvin MK, Abramoff MD. Automated method for identification and artery-venous classification of vessel trees in retinal vessel networks. PLoS One. 2014;9(2):e88061. https://doi.org/10.1371/journal.pone.0088061.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Bell RD, Winkler EA, Singh I, Sagare AP, Deane R, Wu Z, et al. Apolipoprotein E controls cerebrovascular integrity via cyclophilin A. Nature. 2012;485(7399):512–6. https://doi.org/10.1038/nature11087.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Zudaire E, Gambardella L, Kurcz C, Vermeren S. A computational tool for quantitative analysis of vascular networks. PLoS One. 2011;6(11):e27385. https://doi.org/10.1371/journal.pone.0027385.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Di Ieva A, Grizzi F, Jelinek H, Pellionisz AJ, Losa GA. Fractals in the neurosciences, part I: general principles and basic neurosciences. Neuroscientist. 2014;20(4):403–17. https://doi.org/10.1177/1073858413513927.

    Article  CAS  PubMed  Google Scholar 

  14. Di Ieva A, Esteban FJ, Grizzi F, Klonowski W, Martin-Landrove M. Fractals in the neurosciences, part II: clinical applications and future perspectives. Neuroscientist. 2015;21(1):30–43. https://doi.org/10.1177/1073858413513928.

    Article  PubMed  Google Scholar 

  15. Karperien A, Ahammer H, Jelinek HF. Quantitating the subtleties of microglial morphology with fractal analysis. Front Cell Neurosci. 2013;7:3. https://doi.org/10.3389/fncel.2013.00003.

    Article  PubMed  PubMed Central  Google Scholar 

  16. Borodinsky LN, Fiszman ML. A single-cell model to study changes in neuronal fractal dimension. Methods. 2001;24(4):341–5. https://doi.org/10.1006/meth.2001.1204.

    Article  CAS  PubMed  Google Scholar 

  17. Caserta F, Eldred WD, Fernandez E, Hausman RE, Stanford LR, Bulderev SV, et al. Determination of fractal dimension of physiologically characterized neurons in two and three dimensions. J Neurosci Methods. 1995;56(2):133–44.

    Article  CAS  PubMed  Google Scholar 

  18. Gadde SG, Anegondi N, Bhanushali D, Chidambara L, Yadav NK, Khurana A, et al. Quantification of vessel density in retinal optical coherence tomography angiography images using local fractal dimension. Invest Ophthalmol Vis Sci. 2016;57(1):246–52. https://doi.org/10.1167/iovs.15-18287.

    Article  CAS  PubMed  Google Scholar 

  19. Di Ieva A, Grizzi F, Sherif C, Matula C, Tschabitscher M. Angioarchitectural heterogeneity in human glioblastoma multiforme: a fractal-based histopathological assessment. Microvasc Res. 2011;81(2):222–30. https://doi.org/10.1016/j.mvr.2010.12.006.

    Article  PubMed  Google Scholar 

  20. Obenaus A, Ng M, Orantes AM, Kinney-Lang E, Rashid F, Hamer M, et al. Traumatic brain injury results in acute rarefication of the vascular network. Sci Rep. 2017;7(1):239. https://doi.org/10.1038/s41598-017-00161-4.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Honig MG, Hume RI. Dil and diO: versatile fluorescent dyes for neuronal labelling and pathway tracing. Trends Neurosci. 1989;12(9):333–5. 40-1

    Article  CAS  PubMed  Google Scholar 

  22. Konno A, Matsumoto N, Okazaki S. Improved vessel painting with carbocyanine dye-liposome solution for visualisation of vasculature. Sci Rep. 2017;7(1):10089. https://doi.org/10.1038/s41598-017-09496-4.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Okyere B, Giridhar K, Hazy A, Chen M, Keimig D, Bielitz RC, et al. Endothelial-specific EphA4 negatively regulates native pial collateral formation and re-perfusion following hindlimb ischemia. PLoS One. 2016;11(7):e0159930. https://doi.org/10.1371/journal.pone.0159930.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, et al. Fiji: an open-source platform for biological-image analysis. Nat Methods. 2012;9(7):676–82. https://doi.org/10.1038/nmeth.2019.

    Article  CAS  PubMed  Google Scholar 

  25. Okyere B, Creasey M, Lebovitz Y, Theus MH. Temporal remodeling of pial collaterals and functional deficits in a murine model of ischemic stroke. J Neurosci Methods. 2017;293:86–96. https://doi.org/10.1016/j.jneumeth.2017.09.010.

    Article  PubMed  PubMed Central  Google Scholar 

  26. Jullienne A, Salehi A, Affeldt B, Baghchechi M, Haddad E, Avitua A et al. Male and female mice exhibit divergent responses of the cortical vasculature to traumatic brain injury. J Neurotrauma. 2018. https://doi.org/10.1089/neu.2017.5547.

  27. Harb R, Whiteus C, Freitas C, Grutzendler J. In vivo imaging of cerebral microvascular plasticity from birth to death. J Cereb Blood Flow Metab. 2013;33(1):146–56. https://doi.org/10.1038/jcbfm.2012.152.

    Article  CAS  PubMed  Google Scholar 

  28. Kienast Y, von Baumgarten L, Fuhrmann M, Klinkert WE, Goldbrunner R, Herms J, et al. Real-time imaging reveals the single steps of brain metastasis formation. Nat Med. 2010;16(1):116–22. https://doi.org/10.1038/nm.2072.

    Article  CAS  PubMed  Google Scholar 

  29. Fumagalli S, Ortolano F, De Simoni MG. A close look at brain dynamics: cells and vessels seen by in vivo two-photon microscopy. Prog Neurobiol. 2014;121:36–54. https://doi.org/10.1016/j.pneurobio.2014.06.005.

    Article  PubMed  Google Scholar 

  30. Shen Q, Huang S, Duong TQ. Ultra-high spatial resolution basal and evoked cerebral blood flow MRI of the rat brain. Brain Res. 2015;1599:126–36. https://doi.org/10.1016/j.brainres.2014.12.049.

    Article  CAS  PubMed  Google Scholar 

  31. Ostergaard L, Engedal TS, Aamand R, Mikkelsen R, Iversen NK, Anzabi M, et al. Capillary transit time heterogeneity and flow-metabolism coupling after traumatic brain injury. J Cereb Blood Flow Metab. 2014;34(10):1585–98. https://doi.org/10.1038/jcbfm.2014.131.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Sato Y, Nakajima S, Shiraga N, Atsumi H, Yoshida S, Koller T, et al. Three-dimensional multi-scale line filter for segmentation and visualization of curvilinear structures in medical images. Med Image Anal. 1998;2(2):143–68.

    Article  CAS  PubMed  Google Scholar 

  33. Zeller K, Vogel J, Kuschinsky W. Postnatal distribution of Glut1 glucose transporter and relative capillary density in blood-brain barrier structures and circumventricular organs during development. Brain Res Dev Brain Res. 1996;91(2):200–8.

    Article  CAS  PubMed  Google Scholar 

  34. Wang DB, Blocher NC, Spence ME, Rovainen CM, Woolsey TA. Development and remodeling of cerebral blood vessels and their flow in postnatal mice observed with in vivo videomicroscopy. J Cereb Blood Flow Metab. 1992;12(6):935–46. https://doi.org/10.1038/jcbfm.1992.130.

    Article  CAS  PubMed  Google Scholar 

  35. Krafft PR, Rolland WB, Duris K, Lekic T, Campbell A, Tang J, et al. Modeling intracerebral hemorrhage in mice: injection of autologous blood or bacterial collagenase. J Vis Exp. 2012;67:e4289. https://doi.org/10.3791/4289.

    Article  Google Scholar 

  36. Suzuki H, Ayer R, Sugawara T, Chen W, Sozen T, Hasegawa Y, et al. Protective effects of recombinant osteopontin on early brain injury after subarachnoid hemorrhage in rats. Crit Care Med. 2010;38(2):612–8. https://doi.org/10.1097/CCM.0b013e3181c027ae.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Mu J, Ostrowski RP, Krafft PR, Tang J, Zhang JH. Serum leptin levels decrease after permanent MCAo in the rat and remain unaffected by delayed hyperbaric oxygen therapy. Med Gas Res. 2013;3(1):8. https://doi.org/10.1186/2045-9912-3-8.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Hama H, Hioki H, Namiki K, Hoshida T, Kurokawa H, Ishidate F, et al. ScaleS: an optical clearing palette for biological imaging. Nat Neurosci. 2015;18(10):1518–29. https://doi.org/10.1038/nn.4107.

    Article  CAS  PubMed  Google Scholar 

  39. Richardson DS, Lichtman JW. Clarifying tissue clearing. Cell. 2015;162(2):246–57. https://doi.org/10.1016/j.cell.2015.06.067.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Zhang Lin-Yuan, Lin Pan, Pan Jiaji, Ma Y, Wei Zhenyu, Jiang Lu et al. CLARITY for high-resolution imaging and quantification of vasculature in the whole mouse brain. Aging and Disease 2018;9(1). doi: 10.14336/ad.2017.0613.

  41. Hama H, Kurokawa H, Kawano H, Ando R, Shimogori T, Noda H, et al. Scale: a chemical approach for fluorescence imaging and reconstruction of transparent mouse brain. Nat Neurosci. 2011;14(11):1481–8. https://doi.org/10.1038/nn.2928.

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We would like to thank Monica Romero for her assistance with the confocal imaging which was performed in the LLUSM Advanced Imaging and Microscopy Core with support of NSF Grant MRI-DBI 0923559 and the Loma Linda University School of Medicine.

Funding

This study was supported by an NIH Program Project grant from the National Institute of Neurological Disorders and Stroke (1P01NS082184, Project 3).

Author information

Authors and Affiliations

  1. Cell, Molecular and Developmental Biology Program, University of California, Riverside, 1140 Bachelor Hall, Riverside, CA, 92521, USA

    Arjang Salehi & Andre Obenaus

  2. Department of Basic Sciences, Loma Linda University, Loma Linda, CA, 92354, USA

    Arjang Salehi, Amandine Jullienne, Mary Hamer & Andre Obenaus

  3. Department of Anatomy and Neurobiology, University of California, Irvine, Irvine, CA, 92697-4475, USA

    Kara M. Wendel & Andre Obenaus

  4. Department of Pediatrics, University of California, Irvine, Irvine, CA, 92697-4475, USA

    Mary Hamer & Andre Obenaus

  5. Department of Physiology and Pharmacology, Loma Linda University School of Medicine, Loma Linda, CA, 92350, USA

    Jiping Tang, John H. Zhang & William J. Pearce

  6. Department of Anesthesiology, Loma Linda University School of Medicine, Loma Linda, CA, 92350, USA

    John H. Zhang

  7. Department of Neurosurgery, Loma Linda University School of Medicine, Loma Linda, CA, 92350, USA

    John H. Zhang

  8. Center for Perinatal Biology, Loma Linda University, Loma Linda, CA, 92350, USA

    William J. Pearce

  9. Molecular and Integrative Physiology, University of Michigan, Ann Arbor, MI, 48101, USA

    Richard A. DeFazio

  10. Department of Neurology, University of California, San Francisco, San Francisco, CA, 94158, USA

    Zinaida S. Vexler

Authors
  1. Arjang Salehi
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Amandine Jullienne
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Kara M. Wendel
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Mary Hamer
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Jiping Tang
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. John H. Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. William J. Pearce
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Richard A. DeFazio
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Zinaida S. Vexler
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Andre Obenaus
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

AS, AJ, KW, MH, and AO contributed to the conception and design of the study. AS, AJ, KW, and MH acquired and analyzed the data. AS, AJ, and AO drafted a significant portion of the manuscript and figures. AO, JT, JZ, WP, RD, and ZV edited the final draft. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Andre Obenaus.

Ethics declarations

Conflict of Interest

The authors declare that they have no conflict of interest.

Research Involving Animals

All procedures performed in studies involving animals were in accordance with the ethical standards of Loma Linda University Animal Health and Safety Committee (according to the Guide For the Care and Use of Laboratory Animals, Eighth edition) at which the studies were conducted.

Resource Sharing

We will provide published data and relevant protocols upon request. Unpublished information can be made available by providing a request to the Principal Investigator by phone call or email.

Electronic Supplementary Material

ESM 1

(DOCX 6.81 MB)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Salehi, A., Jullienne, A., Wendel, K.M. et al. A Novel Technique for Visualizing and Analyzing the Cerebral Vasculature in Rodents. Transl. Stroke Res. 10, 216–230 (2019). https://doi.org/10.1007/s12975-018-0632-0

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12975-018-0632-0

Keywords

Search

Navigation