The overall goal of these procedures for isolation of extracellular matrix and ex vivo vasodilation is to study IRF5 inhibitory peptide treatment in rodents. This method can help answer how changes in the extracellular matrix can augment or attenuate disease progression. The main advantage of the vasodilation studies is to give us an insight into in vivo processes without having to take all the signaling components into consideration.
The isolation of cardiac extracellular matrix can be applied to other organs such as hind-limb or lung. We first had the idea when we started to focus on the duplication of fibrillin one in the tight skin mouse, and recognized the impact of altered extracellular matrix and cell behavior. After anesthetizing the mouse and preparing it for surgery, open the skin using a pair of scissors.
Cutting up from the chest to lateral to the jawbone with care as to not disturb the underlying tissue towards the front of the jaw. Then dissect the fascialis artery by removing the soft tissue around it. While doing this, be careful to avoid damaging the artery.
Bathe the exposed tissue in mops buffer so they remain healthy. Once the fascialis artery has been fully exposed, grasp the fascialis proximal to the first bifurcation distal to where the artery will be cut. Then, cut it away from the parent artery and take hold of the fascialis artery with the forceps.
Cut the two branches coming off the fascialis and then gently lift the vessel while severing connections to surrounding tissue until the bifurcation is reached. At the bifurcation, cut both daughter arteries to free the vessel and transfer it into a dish of mops buffer where it will remain hydrated until needed. To measure vasodilation, first prepare to cannulate the vessels.
Load glass pipettes with a terminal diameter of 125 to 175 microns full of mops buffer. Also fill solution reservoirs with mops. Now cannulate the isolated vessels.
Tie them onto glass pipettes using opthalmic suture. Be sure to prevent air bubbles from forming in the pipettes. Next, mount the vessel chambers on an inverted microscope with the video camera attachment.
Run the video through the video caliper measurement system for an on screen measurement of the vessels. Now pressurize the vessels using a two step process. First raise the mops filled reservoirs at a height above the vessel for 20 millimeters of mercury of pressure.
Open the line with the mops to the vessel. As the vessel pressurizes look for signs of leaking around the ties and down the length of the vessel. If no leaks are found, raise the reservoirs to produce 60 millimeters of mercury of pressure.
Then inspect the vessels for leaks again. Once the pressure is maintained without leaks, equilibrate the vessel for 30 minutes at this pressure. After being equilibrated, measure vasodilation in response to a compound of interest.
First constrict the vessel by one half to three quarters by adding U46619. Let the solution bathe the vessels for six minutes to stabilize. Then, gradually add the test compound to the bath solution such as acetylcholine.
Every two minutes, increase the concentration of the test solution in the bath by 10 beginning at 000001 molar and progressing to 001 molar. The changes in vasodilation raised questions about signaling. But our focus is on the heart.
To reduce the use of animals, we chose to isolate cardiac extracellular matrix to focus on the signaling initiated from the outside. Begin by collecting the heart from an anesthetized mouse. Transfer it to a beaker loaded with 15 milliliters of hypertonic one percent sodium dodecyl sulfate.
Keep the solution stirring gently on a stir plate at room temperature for 18 hours. The next day, change the solution to 15 milliliters of 5 percent triton X100 for 30 minutes followed by 15 milliliters of PBS for 15 minutes. Then snap freeze the ECM and liquid nitrogen in a 1.5 milliliter microcentrifuge tube.
Once frozen, transfer the tissue into a pre-cooled mortar and pestle and grind it into a powder. Suspend the powder in PBS with added antibiotic at a 1:100 concentration in a viscous solution. Sonicate the suspension for 30 to 60 seconds in an ice bath at a high sonication setting.
ECM protein is now isolated and must stay cold. Before using the protein, measure its concentration using a Bradford Assay. The IRF5D peptide is an inhibitory peptide that differs greatly from normal IRF5.
IRF5D has only 17 amino acids and binds to IRF5's phosphorylation site. This prevents phosphorylation and homodimerization, thus interfering with the formation of the IRF5 active state. Heterozygous tight skin mice were treated with IRF5D subcutaneously for three weeks.
The number of neutrophil and CD64 were decreased as determined by immunohistochemistry in the average number of stained cells per field of view. IRF5D treatments improved acetylcholine induced vasodilation of fascialis arteries in heterozygous tight skin mice treated with IRF5D compared to those treated with phosphate buffer. In cultured myocytes derived from the cardiac tissue of the transgenic mice, there were more IRF5 positive cells in cultures from tight skin heterozygous mice than in control mice.
Treatment of either culture with IRF5D resulted in a reduction of IRF5 positive nuclei. After watching this video, you should have a good understanding of how to decellularize a heart. Once mastered, this technique can be transposed to many organs and aid to answer a variety of questions And remember that it is important not to overdigest the matrix.
You should also have a good understanding of how to dissect and hang the fascialis artery. Once mastered, this technique should take approximately 60 minutes and can be used on just about any vascular bed. It is important to remember how fragile the endothelium is, proceed with caution during the dissection.
It is not uncommon to have healthy smooth muscle and damaged endothelium.
