Skip to main content

Quantitative Analysis of Angiogenesis in the Allantois Explant Model

Abstract

The murine allantois represents a powerful system for the analysis of developmental angiogenesis ex vivo, producing a complex vascular network in less than a day’s time. This can be visualised by appropriate antibody staining. Angiogenesis in the explants occurs in the absence of external, confounding factors. Since the allantois is taken at an early developmental stage, it is a useful system for studying embryonic lethal mutations with no need for breeding conditional knock-outs. In addition, allantois explants are useful for testing inhibitors without any need for animal procedures. Manual quantification of angiogenesis in the complex vascular networks is time and labour intensive. We developed AngioTool, a piece of software which allows the quick, hands-off and reproducible quantification of microscopic images of vascular networks. AngioTool is available free of charge and was developed for analysis of angiogenesis in the allantois explant, although it is also suitable for other systems. Parameters measured include the overall size of the vascular network, average and total vessel length, percentage of area covered by vessels and number of endpoints. In addition, AngioTool calculates the so-called “branching index” (branch points/unit area of the explant), measuring the sprouting activity of an explant whilst correcting for its overall size. The entire experimental protocol including analysis takes ≈5 days to complete.

Keywords

  • Vascular Network
  • Skim Milk Powder
  • Excel File
  • Tissue Culture Incubator
  • Developmental Angiogenesis

These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

This is a preview of subscription content, access via your institution.

Buying options

Chapter
USD   29.95
Price excludes VAT (USA)
  • DOI: 10.1007/978-94-007-4581-0_12
  • Chapter length: 16 pages
  • Instant PDF download
  • Readable on all devices
  • Own it forever
  • Exclusive offer for individuals only
  • Tax calculation will be finalised during checkout
eBook
USD   79.99
Price excludes VAT (USA)
  • ISBN: 978-94-007-4581-0
  • Instant PDF download
  • Readable on all devices
  • Own it forever
  • Exclusive offer for individuals only
  • Tax calculation will be finalised during checkout
Softcover Book
USD   99.99
Price excludes VAT (USA)
Hardcover Book
USD   109.99
Price excludes VAT (USA)
Fig. 1
Fig. 2
Fig. 3

References

  1. Adams RH, Alitalo K (2007) Molecular regulation of angiogenesis and lymphangiogenesis. Nat Rev Mol Cell Biol 8:464–478

    PubMed  CrossRef  CAS  Google Scholar 

  2. Downs KM, Gifford S, Blahnik M, Gardner RL (1998) Vascularization in the murine allantois occurs by vasculogenesis without accompanying erythropoiesis. Development 125:4507–4520

    PubMed  CAS  Google Scholar 

  3. Drake CJ, Fleming PA (2000) Vasculogenesis in the day 6.5 to 9.5 mouse embryo. Blood 95:1671–1679

    PubMed  CAS  Google Scholar 

  4. Frangi AF, Niessen WJ, Vincken KL, Viergever MA (1998) Multiscale vessel enhancement filtering. In: Wells WM, Colchester A, Delp S (eds) Medical image computing and computer-assisted intervention, Lecture notes in computer science. Springer, Berlin, pp 130–137

    CrossRef  Google Scholar 

  5. Fruttiger M (2007) Development of the retinal vasculature. Angiogenesis 10:77–88

    PubMed  CrossRef  Google Scholar 

  6. Lee TC, Kashyap RL, Chu CN (1994) Building skeleton models via 3-D medial surface axis thinning algorithms. CVGIP-Graphical Model Image Process 56:462–478

    CrossRef  Google Scholar 

  7. Manniesing R, Viergever MA, Niessen WJ (2006) Vessel enhancing diffusion: a scale space representation of vessel structures. Med Image Anal 10:815–825

    PubMed  CrossRef  Google Scholar 

  8. Perryn ED, Czirok A, Little CD (2008) Vascular sprout formation entails tissue deformations and VE-cadherin-dependent cell-autonomous motility. Dev Biol 313:545–555

    PubMed  CrossRef  CAS  Google Scholar 

  9. Potente M, Gerhardt H, Carmeliet P (2011) Basic and therapeutic aspects of angiogenesis. Cell 146:873–887

    PubMed  CrossRef  CAS  Google Scholar 

  10. Russell WMS, Burch RL (1959) The principles of humane experimental technique. Methuen & Co.Ltd, London

    Google Scholar 

  11. Sato Y, Nakajima S, Shiraga N, Atsumi H, Yoshida S, Koller T, Gerig G, Kikinis R (1998) Three-dimensional multi-scale line filter for segmentation and visualization of curvilinear structures in medical images. Med Image Anal 2:143–168

    PubMed  CrossRef  CAS  Google Scholar 

  12. Takakura N, Huang XL, Naruse T, Hamaguchi I, Dumont DJ, Yancopoulos GD, Suda T (1998) Critical role of the TIE2 endothelial cell receptor in the development of definitive hematopoiesis. Immunity 9:677–686

    PubMed  CrossRef  CAS  Google Scholar 

  13. Uemura A, Kusuhara S, Katsuta H, Nishikawa S (2006) Angiogenesis in the mouse retina: a model system for experimental manipulation. Exp Cell Res 312:676–683

    PubMed  CrossRef  CAS  Google Scholar 

  14. Zudaire E, Gambardella L, Kurcz C, Vermeren S (2011) A computational tool for quantitative analysis of vascular networks. PLoS One 6:e27385

    PubMed  CrossRef  CAS  Google Scholar 

Download references

Acknowledgements

This work was funded by UK Medical Research Council grant G0700740. SV is a Biotechnology and Biological Sciences Research Council David Phillips Fellow (BB/C520712).

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Sonja Vermeren .

Editor information

Editors and Affiliations

Rights and permissions

Reprints and Permissions

Copyright information

© 2012 Springer Science+Business Media Dordrecht

About this chapter

Cite this chapter

Gambardella, L., Zudaire, E., Vermeren, S. (2012). Quantitative Analysis of Angiogenesis in the Allantois Explant Model. In: Zudaire, E., Cuttitta, F. (eds) The Textbook of Angiogenesis and Lymphangiogenesis: Methods and Applications. Springer, Dordrecht. https://doi.org/10.1007/978-94-007-4581-0_12

Download citation