Abstract
The primary function of the vascular endothelium in vertebrate organisms is to serve as a barrier between the blood and each tissue of the body, whereby the permeability of the endothelium to blood cells, plasma macromolecules, and water can be adapted according to the physiological need. In certain diseases, cytokines and growth factors are released that target the endothelial barrier to transiently increase vascular permeability; however, their prolonged presence may cause chronic vascular hyperpermeability and thereby tissue-damaging edema. The Miles assay is an in vivo technique that allows researchers to study vascular hyperpermeability through the proxy measurement of vascular leakage. Here, we provide a detailed protocol on how to perform this procedure in the mouse, which is the most widely used model organism to study mammalian physiology and pathology. The procedure involves the intravenous injection of Evans blue dye to label the circulating albumin followed by multiple intradermal injections of permeability-inducing agents and vehicle control solutions into opposing flanks of the mouse. Consequently, Evans blue dye gradually leaks into the dermis, where it accumulates and can be extracted for quantification as leakage induced by the permeability-inducing agent relative to the vehicle. The Miles assay can be performed in wild type or genetically modified mouse models and may be combined with drug administration to study molecular mechanisms that regulate vascular permeability and identify agents/targets capable of inducing or blocking hyperpermeability.
Highlights
The primary function of the cardiovascular system is to enable the transfer of gases, nutrients, and waste products between the circulation and tissues in all organs
A clear increase in Evans blue dye leakage in skin samples injected with VEGF-A compared to PBS was apparent in situ (Figure 1E) and after extraction of the dye in formamide (Figure 2A)
Quantification of multiple experiments illustrates that 50 ng of VEGFA significantly induces more Evans blue leakage than PBS alone (Figure 2B), with an average 3-fold increase in vascular leakage compared to vehicle (Figure 2C)
Summary
The primary function of the cardiovascular system is to enable the transfer of gases, nutrients, and waste products between the circulation and tissues in all organs. Certain stimuli cause a partial breakdown of the endothelial barrier to increase the fluid extravasation from the circulation into the interstitium above the basal levels[1] Such hyperpermeability is observed, for example, at sites of tissue trauma, in inflammation, in tumors, during sepsis, in eyes with neovascular disease or in the brain and heart, when tissue ischemia occurs due to a stroke or myocardial infarction, respectively[5,6,7,8]. The Miles assay is a well-established, commonly used and relatively simple technique that measures vascular leakage in vivo as a surrogate measurement of vascular hyperpermeability Even though it does not take into account compounding factors that may increase vascular leakage independently of endothelial barrier regulation, such as blood pressure or blood flow, the Miles assay is generally thought to provide a reliable method to evaluate the permeability-modulating activity of substances and identify the signaling mediators that promote their activity. All animal work was carried out following UK Home Office and institutional Animal Welfare and Ethical Review Body (AWERB) guidelines
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