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In Vivo Vascular Leakage Assay

  • Maria Elena Ferrero
Part of the Methods in Molecular Medicine™ book series (MIMM, volume 98)

Abstract

This chapter describes the methods for measuring the increase in vascular permeability induced by tumor necrosis factor α (TNFα), or other mediators, in vivo in animal models. Mouse liver or kidney are perfused through the portal vein or the renal artery, respectively, by intravascular injection of a blue dye (trypan blue or Evans blue) combined with albumin. When endothelial permeability is increased, the dye extravasates and reaches the subendothelial spaces. Thus, subsequent washing of dye-perfused organs with saline cannot remove the dye. The perfusates are drawn from the suprahepatic inferior vena cava (for the liver) and from the renal vein (for the kidney). After perfusion, the livers and kidneys are removed, homogenized, and centrifuged. Spectrophotometric analysis of supernatants, at 540 nm, is then performed. The increase in optical density values is indicative of the increase in dye retention, hence of vascular leakage.

Key Words

Vascular permeability dye perfusion mouse liver kidney TNFα 

References

  1. 1.
    Wall, P. D. (1993) Vigilance in defence of animal welfare. Pain 54, 239.PubMedCrossRefGoogle Scholar
  2. 2.
    Branster, M. V. and Morton, R. K. (1957) Isolation of intact liver cells. Nature 180, 1283–1284.PubMedCrossRefGoogle Scholar
  3. 3.
    Ferrero, E., Villa, A., Ferrero, M. E., Toninelli, E., Bender, J. R., Pardi, R., et al. (1996) Tumor necrosis factor α-induced vascular leakage involves PECAM 1 phosphorilation. Cancer Res. 56, 3211–3215.PubMedGoogle Scholar
  4. 4.
    Fulgenzi, A., Bertelli, A. A., Magni, E., Ferrero, E., and Ferrero M. E. (2001) In vivo inhibition of TNF alpha-induced vascular permeability by resveratrol. Transplant Proc. 33, 2341–2343.PubMedCrossRefGoogle Scholar
  5. 5.
    Ferrero, E., Zocchi, M. R., Magni, E., Panzeri, M. C., Curnis, F., Rugarli, C., et al. (2001) Roles of tumor necrosis factor p55 and p75 receptors in TNF-alpha-induced permeability. Am. J. Physiol. Cell Physiol. 281, C1173–C1179.PubMedGoogle Scholar
  6. 6.
    Bertelli, A. A. E., Baccalini, R., Battaglia, E., Falchi, M., and Ferrero M. E. (2001) Resveratrol inhibits TNFα-induced endothelial cell activation. Therapie 56, 613–616.PubMedCrossRefGoogle Scholar
  7. 7.
    Ferrero, E., Magni, E., Curnis, F., Villa, A., Ferrero, M. E., and Corti, A. (2002) Regulation of endothelial cell shape and barrier function by chromogranin A. Ann. NY Acad. Sci. 971, 1–4.CrossRefGoogle Scholar
  8. 8.
    Marni, A., Ferrero, M. E., and Gaja, G. (1988) Metabolic function of grafted liver in rats. Transplantation 46, 830–835.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc. 2004

Authors and Affiliations

  • Maria Elena Ferrero
    • 1
  1. 1.Institute of General PathologyUniversity of MilanMilanItaly

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