Measurement of Endothelium-Dependent Vasorelaxation in the Mouse Thoracic Aorta Using Tensometric Small Volume Chamber Myography

We demonstrate the isometric or tensometric myography as the gold standard protocol to measure the reactivity of small resistance or large conduit blood vessels in a well-controlled physiological condition. The measurements are highly reproducible and reliable. The technique is very valuable in allowing researchers to study different layers of blood vessels separately or investigate interaction between different layers of the blood vessel wall simultaneously.

Demonstrating the procedure will be Robert Folk, the research lab specialist from my laboratory. Begin by turning on the water bath and setting it to 37 degrees Celsius. Place two beakers labeled appropriately into the water bath, one with 600 milliliters of HEPES-PSS solution and one with 150 milliliters of high-potassium solution.

Aerate a beaker containing 300 milliliters of HEPES-PSS solution with carbogen gas for at least 10 minutes. Add 30 milliliters of the aerated HEPES-PSS solution to a 50-milliliter centrifuge tube, label appropriately, and place it on ice. Keep the remaining aerated HEPES-PSS solution on ice to be used during the aortic dissection process.

Turn on the four-chamber myograph unit at 5 milliliters of HEPES-PSS solution to each myograph chamber and set the heat to 37 degrees Celsius. Allow the solution in each chamber to be aerated for 30 minutes and check to ensure that the chambers have reached the desired 37 degrees Celsius. Turn on the myograph data acquisition hardware and computer.

Transfer the thoracic cage excised from the euthanized mice to a clear silicone elastomer-coated Petri dish filled with ice cold aerated HEPES-PSS buffer and pin it on both sides. Under the microscope, gently cut and remove the heart and attached aorta from the rib cage and transfer to a clean silicone elastomer-coated dish, then gently remove fat connective tissue and clotted blood from the aorta. Using sharp small scissors, dissect and isolate the whole aorta starting from the arch area to the bottom of the descending aorta.

Cut the dissected aorta into four segments of 2 millimeters each. Use the mini-ruler within the dish as a reference. In chambers already containing 5 milliliters of warmed and aerated HEPES-PSS buffer, carefully slide each of the 2-millimeter aortic segments onto the two mounting pins using forceps.

When placing the chamber back in the myograph unit, slowly move the pins apart by rotating the micrometer counterclockwise so the aortic segment does not slide off the pins. Return each myograph chamber to the unit and start aerating the chambers at 37 degrees Celsius for 20 minutes. Drain the chambers and add 5 milliliters of fresh, warm, aerated HEPES-PSS solution to each chamber.

If needed, readjust the force measurements to read the optimal tension of 6 millinewtons. Rest the tissue for another 15 to 20 minutes. Open the data acquisition software.

Change the tracking numbers from 50 to 1 to 500 to 1 and press Start. Before starting a new experiment, be sure to add the appropriate label on the data acquisition software. Drain the chambers one more time before adding 5 milliliters of high-potassium solution to each chamber.

Drain the chambers again as soon as the contractile response to the high-potassium solution reaches a plateau for force generation. Wash the tissue with HEPES-PSS solution thrice. Add 5 milliliters of high-potassium solution to each chamber.

Drain the chambers as soon as the contractile response to high-potassium solution reaches a plateau for force generation and wash the tissue thrice with HEPES-PSS, then rest the tissue for 15 to 20 minutes. Pre-contract the aortic segments with the vasoconstrictor agent PE at a sub-maximum dose. Allow the PE-induced contraction curve to reach a plateau for tension development.

Add a plateau of tension development to PE at 5 microliters of increasing doses of acetylcholine working stock solutions in 3-minute intervals to establish final concentrations of acetylcholine from 50 picomolar to 10 micromolar in the myograph chamber as described in the text manuscript. After completing the dose response experiment, drain the chambers, wash the aortic segments with warm and aerated HEPES-PSS solution thrice and rest the tissues for 30 minutes. After resting the aortic segments for 30 minutes, drain the chambers and add fresh, warm high-potassium solution to each chamber, then drain the chambers and wash the tissue with HEPES-PSS solution when the contraction response to high-potassium solution reaches an F plateau as demonstrated earlier.

Pre-incubate the aortic segments with L-NAME by adding 10 microliters of the prepared 100-millimolar working stock solution to assess the contribution of nitric oxide production to the observed acetylcholine-induced vasorelaxation. Without removing the L name, add the sub-maximum dose of PE to the chamber to induce aortic contraction. Wait until the PE-induced contraction curve reaches a plateau for tension development, then add acetylcholine to the myograph chamber to achieve a final concentration of 500 nanomolar.

Wait for a few minutes to register any possible changes in force development until the formed plateau is stable. Drain the chambers and wash the tissue with warm aerated HEPES-PSS solution thrice. Position the myograph chamber under the microscope and gently pass a small wire through the lumen of the aorta.

Gently move the wire through the lumen for a short period. Cut the aorta into 2-millimeter aortic rings and mount those rings onto the myograph chamber. Set the optimal tension at 6 millinewtons and allow the tissue to rest for 30 minutes in aerated warm HEPES-PSS buffer, then drain the chambers and add 5 milliliters of fresh warm aerated HEPES-PSS solution to each chamber.

Zero the force for all myograph chambers and slowly rotate the micrometer counterclockwise to increase the distance between the pins until the registered force reaches the desired optimal tension for the mouse aorta. Press the tissue for another 15 to 20 minutes at the optimal tension, then drain the chambers once more before adding 5 milliliters of high-potassium solution to each chamber. Drain the chambers again and wash the tissue with HEPES-PSS solution thrice once the contraction response to the high-potassium ion solution reaches a plateau.

Rest the tissue for 15 to 20 minutes, then pre-contract the aortic segments with PE, the vasoconstrictor agent. When the PE-induced force reaches a plateau, add the sub-maximum concentration of acetylcholine to the myograph chamber to achieve a final concentration of 500 nanomolar. Wait 3 minutes to ensure that the registered plateau for PE-induced force development is not changing before ending the experiment.

The integrity and viability of each isolated vessel were assessed by recording the contractile force generation. The peak of the recorded force was later used to normalize the force generation for the same segment in response to the agonists. The endothelium-mediated vasorelaxation measurements depicted smooth muscle mediated contraction and force generation.

When the PE-induced contraction reached a plateau, increasing doses of acetylcholine achieved the maximum vasorelaxation in the isolated segment. The nitric oxide inhibitor L-NAME completely blocked the acetylcholine-induced vasorelaxation in the pre-contracted aorta indicating that acetylcholine induces aortic vasorelaxation through increasing nitric oxide production. Moreover, removing the endothelial layer from the aortic segments also blocked acetylcholine-induced vasorelaxation underscoring the role of the endothelium in blood vessel relaxation.

Ensure to be gentle while handling, cleaning, and dissecting the blood vessel. It is critical to add the pharmacological agonists and inhibitors with care to not disturb the blood vessel rings mounted onto the myograph chambers. The information can help design experiments to explore potential therapeutic approaches to address the dysfunction of a specific blood vessel's layers.