In vivo vascular leakage assay

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.