This video presents an in vivo assay to test blood vessel permeability. In mice, endothelial cells line the blood vessels and form a semi-permeable barrier. In physiologic conditions, endothelium is impermeable to most proteins so that they are restricted to the blood flow.
In pathologic conditions that promote increased vascular permeability, endothelial cells partially lose contact to each other. Endothelium becomes permeable to small proteins such as albumin. In this assay, we inject intravenously a blue dye that binds to albumin in conditions of increased endothelial permeability.
The D crosses the endothelial barrier and diffuses into the interstitial space. This ultimately results in a bluish coloration of the affected tissue before the experiment is started. Prepare all required materials, pipe it and sterile tips.
Evans blue, sterile spatula, sterile PBS, sterile syringe and needles. Mice, mouse restrainer dissection instruments, dissection board and pins. Balance form ide, heat block for water bath at 55 Celsius degree.
C.Vet spectrophotometer weight Evans blue powder and transfer it to a sterile tube. Add sterile PBS to obtain a 0.5%working concentration. Slowly aspirate 200 microliters Evan Blue solution into the syringe.
Avoid all air bubbles and eliminate any air bubble that escaped in the syringe. Place mouse in a restraint device so that the animal is not really mobile, but its tail can be handled. Place the restraint device on its side, so the lateral tail vein is easily visible and facing upward.
Hold onto the tail with a non-dominant hand between the thumb and the forefinger. Apply pressure at the base of the tail so it works as a tourniquet. Insert the needle at 10 degree angle.
Bevel up and advance into the vein. Keep the needle and the syringe parallel to the tail. Do not apply back pressure to confirm proper placement of the needle in the vein.
As this might collapse the vein slowly inject 200 microliters Evan Blue solution into the tail. Observe the ease with which the plunger advances as this is the proof of correct placement in the vein. If the needle is correctly placed, a small drop of blood appears after removing the needle.
Apply pressure at ejected site until the bleeding stops. Put the mouse back into the cage and observe it for 30 minutes. Sacrifice the mouse and pin it to a whiteboard.
We are presenting in this video both a controlled mouse and the mouse that was genetically engineered to lack a gene that controls endothelial integrity. Open the abdominal and thoracic cavity to expose the organs, collect organs of interest and put them in eend DPH tubes. Before this discarding the mouse carcass.
Make sure you have representative pictures of all organs of interest. Label a new set of tubes, weight an empty tube. Bring the balance value to zero, then transfer the tissue sample to the tube and weight it.
Repeat for all samples. Add 500 microliters for mite to each tube. Transfer all tubes to a 55 Celsius heat block or water bath.
Incubate for 24 to 48 hours to extract Evans blue from all the tissue measure absorbance at 600 nanometer wavelength. Use 500 microliters for IDE as blank. Measure optical density for each sample.
Significant differences in vascular permeability can be easily observed at this step. Calculate nanograms. Evan Blue Extravasated per milligram tissue.
Plot all data in a graph the method they presented, also known as M assay, is used to test vessel permeability in mice before the experiment is started. Tail vein injections should be mastered as this technique requires extensive practice. The results should be recorded as both still pictures and quantitative measurements, and should always be accompanied by molecular analysis.
Vessel permeability is highly dependent on the age and weight of the animal in addition to environmental conditions. Therefore, the experiment should be repeated at least three times and statistical analysis performed.
