The overall goal of this procedure is to induce aortic tissue hypoxia for the initiation of an abdominal aortic aneurysm or AAA with progressive aortic dilation in the rat. This method can help answer key questions in the aortic aneurysms research period about the mechanisms responsible for abdominal aortic aneurysms development and rupture. The main advantage of this technique is that the morphological and the pathological characteristics of that between the rat model are similar to those seen in human abdominal aortic aneurysms.
We first had the idea for this method when we were investigating another possibility of a human AAA. Visual demonstration of this method is critical as the hypoxia-induction surgery is difficult to learn through text-versed instruction alone. Demonstrating the procedure will be Tatsuro Yata, our vascular surgeon, and Hirona Kugo, both past docs from our laboratory.
After confirming the appropriate level of sedation by toe pinch, use an electric razor to shave the abdominal hair of a 300 to 400 milligram rat, followed by a sequential alcohol and povidone-iodine solution disinfection of the exposed skin. Place the rat on the operation table in the supine position and apply ointment to the animal's eyes. Next, use a scissor to make a ventral midline abdominal incision and use sterile gauze and a wound retractor to pack the abdominal contents within the abdominal cavity.
To detach the aorta from the perivascular tissue, use tweezers to gently pick up and tear the retroperitoneum, exposing the aortic wall. Then, exfoliate the infrarenal aorta within the retroperitoneal space from the left renal vein to the bifurcation from the perivascular tissue. Use a 5-0 silk suture to ligate the vessels branching from the abdominal aorta to block the blood supply at a point away from the aorta that does not narrow the aortic lumen.
To block the aortic blood flow, place vascular clips just below the renal artery and just above the bifurcation of the aorta. Similar to creating a dot hemorrhage on the surface of the aortic wall, insert the micro-scissor 5 mm distally from the aortic clip adjacent to the renal artery branches to cut the anterior aortic wall and use a 24-gauge indwelling needle to shallowly insert a polyurethane catheter through the incision. Attach a 1-mL syringe filled with water to the polyurethane and flush the remaining blood from the aorta.
Next, cut a new polyurethane catheter to 10 mm long and completely insert this catheter into the aortic lumen incision. Reposition the mid point of the catheter to match the location of the incision and use an 8-0 monofilament suture to close the incision with an interrupted suture. Ligate the abdominal aorta with a surgeon's knot using 5-0 silk suture and the polyurethane catheter.
When the catheter is secure, remove the vascular clips at the aortic bifurcation and below the renal artery to restore the antegrade blood flow and confirm the presence of gross aortic pulsation. After removing the gauze, use a 4-0 polypropylene suture to tightly close the abdominal incision in two layers to prevent a protruded organ. Then, apply topical lidocaine to the abdominal incision and place the rat on a heating pad with monitoring until full recompense.
Postoperatively, observe the time course development of the enlarged aorta on an ultrasound echogram, measuring the maximum diameter from the inside edge to the outside edge of the abdominal aorta according to standard protocols. 28 days after the surgery, use a scalpel to open the abdomen with a new ventral incision and use scissors to harvest the aneurysm. Place the harvested aortas in 10%neutrally buffered formalin for 24 to 48 hours.
After embedding the aortas in paraffin and obtaining sections of the aortic tissue, according to standard protocols, hydrate the fixed paraffin sections in distilled water for 10 minutes, followed by incubation in Verhoeff's haematoxylin. Rusting and corrosion can be identified with Verhoeff's haematoxylin and van Gieson's solution lab activity. Therefore, the freshness and incubation times of these tissue dyes are important for archiving and optional staining.
After 25 minutes, rinse the sections with three 1-minute washes in water, followed by 10 to 30 dips in differentiating solution. Rinse the sections three times in water again and place the samples in 5%sodium thiosulfate for one minute. After another water wash, immerse the samples in van Gieson solution for five minutes.
Next, dehydrate the tissues in 30-second ascending ethanol immersions, followed by clearance with two 5-minute Xylene incubations. Then use resonance mounting medium to cover the sections with appropriately sized cover slips. In these images, a representative abdominal aortic aneurysm, 28 days post-catheter insertion, is shown.
Measurement of the upper and lower ends of the aorta by transabdominal ultrasonography reveals a normal diameter without dilation immediately after catheter placement, which increases to a maximum size at about 14 days post placement and remains unchanged or slightly increases until the end of the experimental period. Histopathological analysis of the aneurysm on day 28 reveals a prominent degradation of elastic fibers compared to that observed on day zero. Once mastered, this technique can be completed in about one hour if it is performed properly.
Following this procedure, other methods like in vitro analysis can be performed to answer additional questions about the individual serial functions. After its development, this technique paved the way for researchers in the abdominal aortic aneurysm field to explore the disease in specific patient demographics. After watching this video, you should have a good understanding of how to induce aortic tissue hypoxia for the initiation of abdominal aortic aneurysms with progressive aortic dilation in the rat.
Don't forget that working with surgical instruments can be extremely hazardous and that precautions against injury and infection should always be taken while performing this procedure.
