The overall goal of the following experiment is to determine the real-time vascular reactivity of mouse resistant artery in vitro in near physiological conditions. This is achieved by mounting a small segment of the vessel onto two micro cannulas and maintaining it at physiological luminal pressure. The vessel is then constricted with an alpha one receptor agonist phenylephrine to study the ability of any drug to dilate the vessel.
Challenging the constricted vessel with acetylcholine assesses its ability to dilate in real time via an endothelium dependent mechanism. The results show the percentage change in lumen diameter in response to acetylcholine and verify that SGC alpha one deficiency results in impaired endothelium dependent vasodilation. The main advantage of this technique compared to existing methods like wire mamography, is that pressure mamography permits the characterization of small micro vessels, which are more relevant in maintaining blood pressure homeostasis than larger conductance vessels.
This method can help answer key questions in the cardiovascular field, such as hypertension, where macro resistant vessels play a critical role. Start by euthanizing a mouse with pentobarbital. Now open the abdominal cavity and begin dissecting out the mesenteric tissue.
Now identify a third order mesenteric artery and isolate it from the connective and adipose tissue. Differentiating between arteries and veins at the microvessels level is problematic when there is no blood in the vessels. So doing this in the intact animal is preferable.
Next, cut a two to three millimeters long segment of third order mesenteric artery and place it in a dish containing heaps. PSS buffer. The arterial segment should not contain any branching.
Prepare the myo chamber by gently filling the cannulas through inlet and outlet valves with heaps PSS solution From a 10 milliliter syringe, be aware that excessive pressure can damage the fragile transducer connected to cannulas. After filling the cannulas tightly, close both valves. Now transfer the isolated arterial segment into the chamber and mount one end of the vessel onto the right cannula.
Carefully tying it with a fine strand of nylon suture using a syringe flush and fill in the vessel with heap solution via the inlet valve. Now mount the other end of the vessel onto the left cannula and secure the cannula to the vessel with the nylon suture. Fill the chamber with up to 10 milliliters of heap solution.
Check for a leak by gently pushing heap solution via the inlet valve with the help of a syringe. Now place the chamber under the video camera. Start the oxygen and heat the chamber to 37 degrees Celsius while the inlet valve is closed, connected to the P one tube from the first heap solution reservoir.
Let any bubble and solution pass through the tube until there are no bubbles in the tube. Then open the valve. Connect to the left valve of the chamber with tube P two.
Coming from the pressure regulator, make sure that there are no bubbles or leakage in the entire system. Go to the pressure menu on the myo interface panel or in the software and turn on the pump while turning off the flow. Open the program and click collect.
Monitoring and analysis of the vessel lumen vessel diameter, and wall thickness is now possible. To gradually raise the inter luminal pressure, select the P one pressure and enter a sequence of ascending pressure. Values from five to 60 millimeters of mercury vessels, sometimes twist or convolute while the pressure increases, so adjust the tension or strain accordingly with the help of a vertical or longitudinal micro positioner.
Once the pressure is at 60 millimeters and the bath is at 37 degrees Celsius, equilibrate the vessel for at least 45 minutes and up to one hour during the equilibration period. Change the bath solution once with prewarm heaps. Now that the arterial section is equilibrated, apply 10 milliliters of potassium chloride depolarizing solution at 37 degrees Celsius.
The smooth muscle will fully depolarize and achieve maximum constriction. After obtaining a stable constriction, rinse the bath with heap solution three times at 10 minute intervals. Now check the viability of the vessel and integrity of the endothelium.
First, pre constrict the vessel by adding phenylephrine to tend to the negative five moles per liter. When a stable constriction has been obtained, perform a cumulative concentration vasodilation response curve by sequential addition of increasing doses of acetylcholine from 10 to the negative, nine to 10 to the negative five moles per liter after the last acetylcholine dose. Rinse the preparation with heaps three times at 10 minute intervals.
Now, determine the passive lumen diameter by applying calcium free PSS containing two millimoles of EGTA per liter from the recorded tracings. Measure the lumen diameter for each dose response, which will be used to calculate all parameters. Express the acetylcholine relaxation response as a comparison between the treatment with the agonist and calcium free buffer in terms of how much each altered the lumen diameter from the phenylephrine induced constriction.
State pressure Myography was used to study vascular relaxation in response to acetylcholine in mesenteric resistance arteries isolated from wild type and SGC alpha one knockout mice on the S six and black six backgrounds and pre constricted with phenylephrine. SGC alpha one deficiency was associated with the decreased ability of acetylcholine to induce vascular relaxation regardless of the genetic background of the mice studied. However, endothelium dependent relaxation of SGC Alpha one null mice was more markedly impaired in the S six background than the black six Background.
Together these findings suggest that the decreased sensitivity of the vasculature to endothelial dependent relaxation may contribute to the strain specific hypertension in SGC Alpha one knockout mice Once mastered, this technique can be done in one or two hours if it is performed Properly. In addition to this procedure, other methods like confocal microscopy can be performed concurrently on the life blood vessels. This would, for example, allow for the measurement of intracellular iron concentrations that can help identify underlying signaling mechanisms.
