Here, we present a protocol to measure the vascular leakage induced by intradermal administration of permeability promoting agents into the murine skin. This technique can be used to determine the ability of molecules to promote or inhibit vascular leakage or to study the molecular mechanisms that regulate vascular permeability.
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.
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. Blood vessels have organ-specific levels of basal permeability to permit such exchanges1. For instance, blood vessels in the kidney are highly permeable, whilst the blood brain barrier forms a tight, highly impenetrable interface2,3,4. The endothelial cells that form the inner lining of blood vessels provide a physical barrier between the circulation and underlying tissues and regulate vascular permeability in an organ-specific manner. However, certain stimuli cause a partial breakdown of the endothelial barrier to increase the fluid extravasation from the circulation into the interstitium above the basal levels1. 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, respectively5,6,7,8. When chronically elevated, hyperpermeability leads to edema, which in turn causes tissue damage, such as loss of vision in eye disease9. Thus, modeling the vascular hyperpermeability response is desirable for understanding the mechanisms that increase endothelial permeability and to test the efficacy of agents designed to inhibit this.
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. Accordingly, the Miles assay has been integral to numerous studies that have identified mediators of vascular hyperpermeability and their mechanisms of action10,11,12,13,14,15, such as the vascular endothelial growth factor VEGF-A that was originally identified as the vascular permeability factor VPF16.
Originally developed by Miles and Miles to study vascular permeability in guinea pigs17, their assay was subsequently adapted to using mice, which are now the model organism of choice to elucidate molecular mechanisms of vascular permeability regulation due to their exquisite suitability to genetic manipulation. Briefly, Evans blue dye is intravenously injected into adult mice and allowed to circulate for 30 minutes (Figure 1). Permeability-inducing agents versus vehicle control are then intradermally injected in multiple sites on opposing flanks of the mouse to induce vascular leak (Figure 1). Consequently, albumin-bound Evans blue dye extravasates and accumulates in the dermis (Figure 1). After humanely culling the mouse, Evans blue is extracted from the dermis and the level of vascular leak calculated as a ratio of test substance- to vehicle-induced optical density (Figure 2).
Since its first description in 195217, the Miles assay has provided researchers with a relatively quick, simple, and reliable method for studying the molecular mechanisms of vascular hyperpermeability. For example, the Miles assay has been used to test the ability and/or potency of different agents to induce hyperpermeability19,20,21 or to test the efficacy of hyperpermeability blocking agents8,22,23. As another example, agents that induce vascular permeability have been tested in genetically modified mice and their littermate controls to determine the requirements of specific receptors and signal transducing proteins for ligand-induced hyperpermeability responses10,11,12,13,14,15,20,23. Several modified versions of the Miles assay have since been used, for example with respect to the tracer used. Thus, Evans Blue has been substituted in some studies with fluorescence-labeled dextrans of different sizes or microspheres11,13.
The main advantage of the Miles assay over other assays for investigating vascular hyperpermeability mechanisms is that it is comparatively easy to perform and does not require expensive equipment. Furthermore, as an in vivo technique, this assay models vascular leakage in the context of intact blood vessels, as opposed to measuring leakage through in vitro assays such as trans well flux assays or trans endothelial electrical resistance (TEER) assays, which focus, solely, on the endothelial monolayer. The alternative in vivo assays of vascular hyperpermeability may differ from the Miles assay described here by utilizing a different delivery route for the hyperpermeability-inducing agent or by analyzing vascular leakage at different sites, for example by systemic delivery of an agent via the tail vein followed by vascular leakage examination in the lungs or the trachea11,13,15. One limitation of the Miles assay is that the intradermal injection of certain agents may, in principle, also influence blood pressure and flow in addition to endothelial barrier disruption and, therefore, influence vascular permeability also indirectly. Nevertheless, this assay has recently been shown to measure vascular permeability induced by VEGF-A independently of effects on systemic blood pressure15. Another limitation of the Miles assay is that vascular beds in different tissues may respond differently to permeability-promoting agents, and results obtained with a Miles assay in the skin may, therefore, not be representative, for example, of what occurs in the lung or brain.
Animal's age and weight may influence the leakage observed in the Miles assay. To minimize the effect of these variables, researchers should use littermates or similarly sized and aged mice as controls. When assessing mouse mutants for a particular gene of interest, mice should be generated through a heterozygous versus heterozygous breeding strategy and wildtype littermates used as controls. Moreover, it is advisable to use quickly reversible gas anesthetics, such as isoflurane, to avoid vasoconstriction, which has been described for some anesthetic drug administered via parenteral injections24,25. The injection of Evans blue dye into the mouse lateral tail vein is a critical step in this protocol and can greatly influence the quality of the experiment and data. Thus, researchers must be competent in performing tail vein injections and will likely need prior experience or practice before beginning the Miles assay experiment. Alternatively, researchers could use other intravenous routes to deliver Evans blue, such as retro-orbital injection if they are experienced with it. Intradermal injection of permeability-promoting solutions can cause agent-independent damage to the skin. Therefore, intradermal injections should not exceed 20 µl in volume, should always be carried out in the presence of the histamine inhibitor and normalized to vehicle control injections.
The Miles assay has been used by many researchers to evaluate components of the VEGF-A signaling pathway, for example, because VEGF-A-induced vascular permeability causes ascites in cancer patients16 and vision-impairing edema in several neovascular eye disease7. Thus, we and others have used the Miles assay to compare VEGF-A induced vascular leakage in mice that have been genetically modified to lack specific components of the VEGF-A signaling cascade12,13,14,15,20,23. Our recent study using this approach revealed that the main pathological VEGF-A isoform, VEGF165, signals through a complex of VEGFR2 and NRP1, in which the NRP1 cytoplasmic domain promotes the ABL kinase-mediated activation of SRC family kinases to evoke a hyperpermeability response14. VEGFA also promotes blood vessel growth and could, therefore, be used therapeutically to restore blood flow to ischemic tissues if its hyperpermeability activities could be specifically inhibited. Research elucidating the molecular mechanism that steers VEGF-A responses of vascular endothelial cells towards vascular hyperpermeability might, therefore, identify pathways that can be selectively manipulated to inhibit pathological VEGF-A induced edema in diseases, such as cancer or ischemic eye disease. The Miles assay will undoubtedly continue to be a helpful method to underpin these and many other types of functional studies to elucidate molecular regulators and effectors of vascular hyperpermeability.
The authors have nothing to disclose.
We thank Laura Denti, Valentina Senatore and the staff of the Biological Resources Unit at the UCL Institute of Ophthalmology for help with mouse husbandry and Camille Charoy for technical support. This work was supported by a Medical Research Council grant (MR/N011511/1) to C. Ruhrberg and a British Heart Foundation PhD studentship to J.T. Brash (FS/13/59/30649).
Pyrilamine maleate salt | Sigma-Aldrich | P5514-5G | Resuspend in PBS |
Deionised Formamide | Sigma-Aldrich | S4117 | Use in fume cupboard |
Microlance Needles 30g x 0.5" | BD | 305106 | |
Sterile Plastipak 1ml Luer | BD | 309659 | |
Sterile MicroFine syriinges 0.3 ml – 8 mm – 30G | BD | 324826 | |
Evans blue | Sigma Aldrich | E2129 | |
Mouse restrainer | |||
Heat chamber | |||
Hair clippers | |||
Isoflurane | Merial | ap/drugs/220/96 | |
Scalpal | |||
Blunt scissor | |||
Forceps | |||
Eppendorf tubes | |||
Heatblock | |||
Cork board | |||
Benchtop refrigerated centrifuge | |||
Recombinant Vascular Endothelial Growth Factor 165 | Reliatech | M30-004 |