Restenosis following cardiovascular procedures (bypass surgery, angioplasty, or stenting) is a significant problem reducing the durability of these procedures. An ideal therapy would inhibit smooth muscle cell (VSMC) proliferation while promoting regeneration of the endothelium. We describe a model for simultaneous assessment of VSMC proliferation and endothelial function in vivo.
The overall goal of this procedure is to create an animal model that allows the simultaneous assessment of vascular smooth muscle cell proliferation, and reendothelialization. This method can help answer key questions in the field of vascular biology such as what is the mechanism of restenosis after vascular interventions? This technique allows the simultaneous in vivo assessment of vascular smooth muscle cell proliferation and reendothelialization.
Both key events that potentiate or attenuate restenosis after vascular interventions. Demonstrating the procedure will be Dr.Dan Yu, a post doctoral fellow from my laboratory. After confirming a lack of response to toe pinch, remove the hair on the ventral abdomen of an anesthetized, 10 to 12-week old male, C57BL/6 mouse from the sternum to the inguinal region.
Next disinfect the shoulder area with 70%ethanol and use a 25-gauge needle or smaller to administer an appropriate analgesia. Then place the animal in the supine position on a surgical tray heated by an isothermal pad. Scrub the surgical sight with betadine or chlorhexidine and an alcohol rinse.
Repeat both steps two additional times. Apply ointment to the animal's eyes. After making a median abdominal laparotomy incision, reflect the small bowel and duodenum laterally to the right.
Using a new cotton-tipped applicator, expose the retroperitoneum and the abdominal aorta from the left renal vein to the aortic bifurcation. Then use another sterile cotton-tipped applicator to deliver 30 consecutive, five-second crushes to the aorta. After the last crush injury has been administered, remove the cotton roll and allow the viscera to return to their native positions.
Then use a braided, absorbable suture to close the fascia followed by closure of the skin with a running 6-0, nonabsorbable, monofilament suture and place the animal in a recovery cage with clean bedding with monitoring until full recumbency. To analyze the endothelial integrity of the injured cardiac tissue, at the appropriate experimental time point insert a 21-gauge needle into the left ventricle of the exsanguinated heart. Inject PBS until the effluent from the right atrium is clear then perfuse the animal with five milliliters of 0.3%Evans blue dye at physiological pressure over five minutes followed by five milliliters of fresh PBS.
At the end of the PBS infusion, open the abdomen with a midline incision and mobilize the small intestine to the right side of the abdomen, as just demonstrated, to expose the infrarenal aorta. Sharply dissect the aorta from the adjacent tissues and excise the vessel from the left renal vein to the aortic bifurcation for storage in 4%paraformaldehyde. At the end of the fixation, open the aorta longitudinally and pin the tissue to a wax sheet exposing the entirety of the luminal surface for the qualitative assessment of the endothelial integrity by the degree of Evans blue dye.
Crush injury induces aortic wall thickening compared to the aortas of animals treated with a sham procedure with the greatest wall thickness observed three days after injury. The injury also induces a rounded appearance in the cells with a corresponding irregular contour of the lumenal surface. EdU incorporation indicates that the increased wall thickening of the injured aorta is at least in part mediated by the increased proliferation of medial vascular smooth muscle cells.
Phospho-Erk1/2 expression is observed at over 200%greater than the baseline expression three days after the injury with a decreased expression of p27Kip1 measured at early time points. Both phospho-Erk1/2 and p27Kip1 expression return to baseline levels one month after injury suggesting that the observed thickening of the aortic wall is due also in part to increased medial proliferation. As assessed by scanning electron microscopy, crush injury of the luminal surface of the aorta results in the disruption of the endothelial cells and a partial denudation of the endothelium.
Further, damage to the barrier function of the endothelium permits staining of the basement membrane with Evans blue dye while the endothelium of mice treated with a laparotomy alone maintains its barrier function and its white appearance. Once mastered, this technique can be completed in 20 minutes if it's properly performed. While attempting this procedure, it's important to remember to be consistent with the amount of force applied to the aorta and to blind the operating scientist to the experimental condition of the mouse to avoid bias.
Don't forget that working with Evans blue dye and paraformaldehyde can be hazardous and that precautions such as wearing gloves should always be taken while using these materials.
