Evaluating Vascular Hyperpermeability-inducing Agents in the Skin with the Miles Assay

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.

Transcript

The overall goal of this procedure is to measure vascular leakage in the mouse dermis which is induced by intradermal injection of permeability promoting agents. This procedure can help answer key questions about the regulation of vascular permeability in vivo such as which molecules can stimulate or inhibit vascular leakage and what are the signaling pathways involved. The main advantage of this procedure is that it is quick and relatively simple and it mostly uses common laboratory reagents and materials.

The implications of this technique extend towards therapy of diseases with pathological edema. In particular, researchers can use this assay to identify novel molecular targets and to test relevant drugs for their ability to inhibit vascular permeability in a relatively quick and simple manner. Prepare separate solutions of the histamine inhibitor Pyrilamine Maleate and the Evans Blue Dye both in 0.9%saline solution.

Then working in a laminar flow cabinet, sterilize the solutions by passing through a 0.22 micrometer filter. Load the two solutions in separate one milliliter syringes fitted with 30 gauge needles. Next, scruff the mouse.

Then tilt the mouse such that the head is directed towards the ground and the abdomen upwards. After the mouse is positioned, administer 10 microliters per gram of body weight of Pyrilamine Maleate solution by intraperitoneal injection. Next, in order to promote vasodilation, place the mouse in a heat chamber at 37 degrees Celsius for 10 minutes.

Then move the mouse to a mouse restrainer. Next, rub the tail with 70%ethanol for further vasodilation. Administer 100 microliters of Evans Blue Dye solution intravenously through the tail vein.

To prevent bleeding, quickly hold the tail between a finger and the thumb and apply pressure at the site of the injection. Intravenous injection of Evans Blue Dye into the tail vein of the mouse is a difficult and critical step within this procedure. Accordingly, it is essential that researchers are competent in performing this injection before proceeding with the rest of the procedure.

Establishing competence will require prior practice. Use sterile solutions of phosphate buffered saline to dilute VEGF reagent such that the final volume of the dose is 20 microliters. Then load the VEGF permeability inducing reagent and the vehicle control in two separate 300 microliter syringes fitted with 31 gauge needles.

Maintain the needle at 15 degrees angle with the skin and inject 20 microliters of VEGF intradermally into the mouse flank. Locate a raised bubble within the skin to validate successful injection. Perform intradermal injections at two additional sites at least one centimeter apart.

Then turn the mouse to expose the second flank and repeat triplicate injections with the vehicle solution. Maintain a record for each of the injection sites on paper and the time when the injection procedure is terminated. Then transfer the mouse to the home cage.

Continue monitoring the mouse until it regains consciousness and starts moving around the cage. Let the VEGF solution act for 20 minutes before calling the mouse. At this stage, sequentially inject VEGFA and vehicle in all the mice following the same procedure.

After 20 minutes, position each euthanized mouse on its back and pin its feet on a corkboard wrapped in a clean tissue. Next, with the help of blunt scissors, make a three to four centimeter long vertical incision from the lower abdomen to the chest of the dead mouse. Then use forceps and scalpel to tease the skin from both sides of the flank.

Use the scalpel to remove any fat if present in the regions around the leakage site. Then use the forceps and the scalpel to excise the skin regions with leaked Evans Blue Dye. Excised skin regions must be of similar size.

Transfer the skin samples to 1.5 milliliter tubes and label accordingly. Dry the sample by placing the open tube in a heat block overnight. After drying is complete, add 250 microliters of deionized formamide to the sample in a fume hood.

Close the lid of the tube and placed it in a heat block overnight to extract the Evans Dye. Centrifuge the samples at 10, 000 g or higher for 40 minutes in a benchtop centrifuge. After the centrifugation, transfer 100 microliters of each dye-containing supernatant from the centrifuged sample into the wells of a 96-well plate operating in a fume hood.

Read the Evans Blue absorbance using a spectrophotometer. To study vascular leakage induced by VEGF, the mouse is injected with 50 nanograms of VEGFA in phosphate buffered saline or only phosphate buffered saline as vehicle. The image shows an increase in the accumulation of leaked Evans Blue Dye in the skin samples injected with VEGFA compared to the vehicle control in situ.

This 96-well plate image shows the loaded Evans Blue Dye extracted in formamide from the skin sample. This graphical plot displays the normalized absorbance values of paired VEGFA and vehicle-only control samples. The plot shows that injecting 50 nanograms of VEGFA significantly increases the leakage of Evans Dye in comparison to the vehicle only.

This graph is obtained after quantifying the fold changes in the optical density for the VEGFA versus the vehicle samples. The plot shows an average three-fold increase in the vascular leakage after administering VEGFA in comparison to vehicle only. Once mastered, the main segment of this technique can be done in two hours if performed properly.

Whilst performing this procedure, it is important to normalize agent-induced vascular leakage to vehicle-induced vascular leakage so as to account for nonspecific vascular leakage and variation between mice. This procedure can be combined with other methods such as drug administration in order to answer questions such as whether a protein can be targeted to inhibit vascular permeability. After its development, this technique paved the way for researchers in the field of vascular biology to explore the molecular mechanisms of vascular hyperpermeability in genetically modified mice identifying key signaling components that are required for vascular leakage.

After watching this video, you should have a good understanding of how to induce and measure vascular leakage in the mouse dermis.

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Journal (Biology)

Journal (Biology)

Evaluating Vascular Hyperpermeability-inducing Agents in the Skin with the Miles Assay

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