The overall goal of this procedure is to sterly, isolate, and excise the murine aorta. This is accomplished by first exposing the thoracic and abdominal cavity. The second step is to perfuse with cold, sterile saline.
Next, the organs of the thoracic and abdominal cavities are removed, leaving the heart attached to the aorta. The final step is to excise the perivascular adipose tissue and advent tissue from the aorta. Ultimately, a dissecting microscope is used to verify all surrounding tissue is removed, and the aorta can be used for a variety of experimental analyses.
So this technique is helpful in answering key questions in the cardiovascular research field, such as aneurysm development and the formation of atherosclerotic plaques. And so you can look at those by looking at changes in the molecular biology, such as gene and protein expression. After euthanizing the mouse, verify the euthanized state by a toe pinch.
Next, sterilize the skin by wetting the abdominal fur with 70%ethanol. Then secure the mouse at the surgical board supine with a appendages outstretched. Begin the surgery by using forceps to locate and isolate the abdominal skin just inferior to the xiphoid process.
Next, lift the skin up and trim it off with scissors. This should expose the superior portion of the peritoneum and inferior thoracic cavity. Then using forceps and scissors, lift the xiphoid process and make lateral incisions just inferior to it along the subcostal margins.
To dissect the thoracic cavity, enter via the diaphragm, being careful not to damage the heart or any major vessels in the process. Now cranial extend the lateral incisions along the subcostal margins. Thus, remove the anterior portion of the rib cage.
A blunt dissection to remove the heart from the anterior chest wall may be required to complete these incisions. Now using gauze, clean out the chest cavity of excess fluids to make the organs easier to access. Then remove the lungs using forceps and scissors lobe by lobe.
The heart and aorta should then be fully exposed and easily accessed to collect blood via a cardiac puncture. Do so now before proceeding with the perfusion to perfuse the heart, fill a 10 cc syringe with 10 milliliters of sterile ice cold one XPBS. Then attach a 25 gauge needle.
Carefully insert the needle into the left ventricle. Then make an incision in the right atrium so excessive pressure does not build up in the circulatory system in the next step. Now, over the next two to three minutes, slowly eject the PBS from the syringe into the heart.
During the PBS ejection, use sterile gauze to absorb fluid that will pour out from the atrium at the incision. When the PERFUSE eight is all ejected, absorb any remaining fluid with gauze that obscures a clean view of the thoracic cavity. To expose the GI contents, cut coddly through the abdominal wall, extending the incision to the supra pubic area.
Once there cut bilaterally towards the lower limbs to make a skin flap, which can be pinned down or excised. Moving on removes several organs, the lobes of the liver, the pancreas, the spleen, intestines, and lower esophagus. This opens the view to the aorta.
Be extra careful while dissecting the perren region as there are superficial branches from the aorta to the renal arteries in this region. Do this dissection precisely and with care, because the bacteria of the GI can contaminate the other tissues, rinse out the cleared area with one XPBS and soak up excess solution with gauze, the aorta should be easily accessed. So with scissors and forceps, separate the aorta from the spine.
A blunt dissection is appropriate, and use of a dissecting microscope is strongly recommended. The aorta can be removed, beginning coddly or cran. What matters is an orderly plant process of cleaning and separating tissues.
When dissecting the perivascular adipose tissue, remove it using fine micro scissors to avoid damage to the aortic wall. This minimizes the chance of fibroblast contamination when culturing aortic smooth muscle cells. Careful excision of perivascular, adipose tissue and adventitia is very important.
Any leftover tissue could cause contamination in cell culture, but also bias your molecular assays. Using this procedure, an intact aorta originating from the heart descending into the thoracic and abdominal cavities. With the renal arteries still attached is obtained, the aorta can be imaged in situ to quantify morphometric changes, which are diagnostic in the study of abdominal aortic aneurysms.
Subsequently, the aorta can be removed, fixed and stained to look at histological changes such as with hemat, toin and eoin stain, or with ver Hoff Van Geen staining to look at the elastin bands and thus view the structural integrity of the aorta. The aortic cells can also be used for primary cell isolation and for in vitro studies such as viability studies and protein localization. Following the procedure, you could use other techniques such as protein and RNA isolation to look at protein expression, enzyme activity, as well as gene expression.
