The overall goal of this surgical procedure is to create a highly reproducible murine model of arterial denudation injury in the infrarenal abdominal aorta to examine arterial re-endothelialization. This method can help answer key questions in vascular biology. For example, what are the molecular mechanisms involved in endothelial regeneration, and what is the contribution endothelial cell progenitors in the process of repair of a wounded endothelium?
This technique is a unique and practical tool for imposing denudation injury in mouse transgenic models. Gaining insight into the molecular mechanisms of re-endothelialization will aid in preventing thrombotic events following stent deployment in coronary artery and peripheral vascular disease. Though this method can be used to provide insight into the healing arterial denudation injury, it can also be used to investigate other methods of vascular injury repair.
To begin this procedure, place the anesthetized mouse in the supine position on the heated surgical platform and cover its face with the nose cone. Then secure all the extremities with tape and carefully position it so that the abdomen is visible under the stereo microscope. After that, place the sterile adhesive dressings along each edge of the prepared surgical area.
Now, make a three-centimeter incision down the midline of the animal's abdomen, using a scalpel, starting approximately 0.5 centimeters inferior to the xiphoid process. Gentry retract the skin with forceps and remove the skin from the abdominal wall by cutting the fine connective tissue. Apply 0.5%Bupivacaine to the muscle wall, then make a two-centimeter incision on the abdominal wall to expose the abdominal organs.
After that, gently lift the intestines using saline-soaked cotton-tipped applicators, being careful to avoid blunt trauma to the jejunal and ilial arteries. Place them on a warm, sterile saline-soaked gauze sponge outside the abdominal cavity. Then cover the intestines with another warm, sterile saline-soaked gauze sponge to avoid moisture loss.
Next, place a retractor to lateralize the rectum and expose the retroperitoneum. At the level of the inferior pole of the right kidney, use sharp dissecting forceps to make a retroperitonotomy lateral to the aorta, being careful not to injure the inferior vena cava or the surrounding vessels. Then, bluntly dissect the retroperitoneal tissue off the aorta.
Place the vascular clamp over the aorta for at least one minute or other specified time and verify occlusion by visually observing the lack of pulsatility in the distal aorta. Following that, remove the vascular clamp and verify hemostasis. Confirm extravasation by adding 0.5 milliliters of saline into the abdominal cavity and assessing if the saline becomes increasingly blood-tinged.
In this case, no extravasation is noted. Remove any gauze pads placed to aid in visualization of the retroperitoneum and place the intestines back into the abdomen. Subsequently, irrigate the abdominal cavity using pre-warmed sterile saline.
In this procedure, close the abdominal wall muscle layer using a 5-0 braided absorbable single-running suture. Close the skin with one to three drops of polymer adhesive, and then close it with wound clips once the adhesive is set. Afterward, Carprofen is administered and the mouse is transferred to a recovery cage on a heating pad with food and water on the floor.
Closely monitor the mouse for signs of respiratory distress, and administer Carprofen daily for 48 hours post-operatively. Do not leave the animal unattended until it has regained sufficient consciousness to maintain sternal recumbency. Subsequently, return the mouse to a normal cage with food and water.
At any point after the surgery, the mouse can be sacrificed for evaluation. Using a dissecting stereo microscope, carefully separate the intact abdominal aorta from the surrounding tissues. Dissect the adherent connective tissue off the abdominal aorta, then open the aorta via a longitudinal incision along the dorsal surface.
Subsequently, pin the aorta flat on a 35-millimeter silicone-coated dish with the luminal side up for fixation. Subsequently, the tissue can be either embedded for sectioning or used in onfas whole-mount immunocytochemistry. Here, histological evaluation of an uninjured aorta shows the intact tunica intima.
In contrast, clamping of the aorta for ten seconds effectively removed the tunica intima with moderate damage to the smooth muscle cell layer, as demonstrated by H and E staining. Denudation of the endothelium occurs at both 10 seconds and 10 minutes, although to different degrees. To determine the optimal aortic clamping time interval required for complete arterial denudation, mice underwent clamping for 10 seconds, one minute, and 10 minutes, and were immediately sacrificed for analysis, as described in the protocol.
The area of denudation increased with increasing clamp timings. We deemed one minute of aortic clamping sufficient, which produced a highly reproducible area of denudation measuring 0.88 square millimeters, on average, to result in near-complete arterial denudation injury without evidence of ischemic injury. Measurement of the fibrinogen staining 24 hours after injury of six aortas is approximately 0.81 square millimeters, statistically similar to the 0.88 square millimeter injury noted immediately after injury.
Compared to the uninjured endothelium, the wound margin, 24 hours after injury, shows underlying fibrinogen staining, marking re-endothelialization into the denudation injury. Once mastered, this technique can be safely performed within 30 minutes, with ease. By varying the clamp type, application time and area, this technique offers tremendous versatility in denudation injury size that may be manipulated to fit one's study aims.
Following this procedure, immunocytochemistry can be performed to suit one's study aims, such as investigating the proliferative potential of endothelial cells during re-endothelialization, or the contribution of inflammatory cells to vascular injury repair. After this development, this technique paved the way for researchers in the field of vascular biology to explore the role of re-endothelialization in vascular injury repair for transgenic mouse models. After watching this video, you should have a good understanding of how to create a reproducible murine model of arterial denudation injury in the infrarenal abdominal aorta.
