Advanced Abdominal Aortic Aneurysm Modeling in Mice by Combination of Topical Elastase and Oral ß-aminopropionitrile

Animal protocols are approved by the University of Wisconsin-Madison Institutional Animal Care and Use Committee (M005792).

1. Animal maintenance

1. Raise mice on standard maintenance chow. Use adult mice or young adult mice (8-12 weeks old).
   NOTE: Using adults ensures that the animals have reached full maturity and limits any chance that changes in aortic diameter could be related to animal growth. For this study, we utilized male and female C57BL/6J mice aged 22-24 weeks at the time of surgery. Lu and colleagues did not observe significant differences in the aneurysmal response between younger and older mice⁸. Additionally, while most aneurysm modeling is performed in male mice, this model successfully induces AAAs in both male and female mice⁹.
2. Determine the study duration and assign animals to treatment or sham (control) groups. Administer 0.2% BAPN drinking water to treatment group mice and subject them to surgery with topical application of active elastase to the infrarenal aorta. Administer untreated water to control animals and subject them to surgery with the application of denatured elastase to the infrarenal aorta.

2. Initiation of B-aminopropionitrile (BAPN)-supplemented drinking water

1. Two days before surgery, start treatment mice on 0.2% BAPN drinking water. Prepare BAPN water in larger volumes and store it in the dark at 4 °C for up to 28 days. Be sure the BAPN water reaches room temperature before giving it to the mice.
   NOTE: We recommend BAPN water be replaced in cages every 7 days throughout the duration of the study.

3. Day of surgery material preparation

1. Cut surgical gloves into 5 mm x 10 mm strips, which will be used later to help isolate the aorta before treatment with elastase. Prepare a surgical drape by cutting a ~ 1.5 cm x 3 cm sized oval in the center of a surgical drape. Unfold the 2 in x 2 in gauze and cut in half to create strips of gauze roughly 2.5 cm x 10 cm to be used later for retraction of abdominal contents. Autoclave all surgical instruments (see the Table of Materials) and set up a sterile surgical field as shown by the example in Figure 1.

Figure 1: Example of the sterile surgery setup in preparation for the elastase/BAPN murine model of AAA. Abbreviations: BAPN = ß-aminopropionitrile; AAA = abdominal aortic aneurysm. Please click here to view a larger version of this figure.

2. Prepare a postoperative recovery cage by placing a clean cage under a heat lamp and place saline near the lamp to warm to body temperature (37 °C). Ensure the heat lamp is safely positioned so that the recovery cage and saline are warm but are not beyond 37 °C. Turn on the water pump to begin circulating warm water through the heating pad.

4. Animal preparation for surgery

1. Place the mice in an induction chamber and anesthetize them with 5% isoflurane at 200 mL/min using a low-flow electronic vaporizer. While anesthetized, weigh each mouse and deliver 0.6 mg/kg of buprenorphine ER and 20 mg/kg of carprofen subcutaneously for analgesia. Use electric hair trimmers to clip the fur on the abdomen, from the lower abdomen to inferior of the xyphoid process. Use gauze or a laboratory wipe to brush away excess hair. Return mice to their cage and wait at least 20 min for analgesia to take effect before proceeding with surgery.
2. After at least 20 min since analgesia was administered, place the mouse in an anesthesia induction chamber and again deliver 5% isoflurane at 200 mL/min using a low-flow electronic vaporizer until the mouse is sedated.
3. Remove the sedated mouse from the induction chamber and place it supine on the surgical field. Apply eye gel and secure the nose cone with surgical tape. Reduce the delivered inhaled isoflurane to a maintenance rate of 1-2% at 50 mL/min. Secure the mouse's front and hind paws with surgical tape.
4. Examine the mouse's lower abdomen for the bladder. Gently apply external pressure to the bladder between the thumb, index, and middle fingers to induce voiding; meanwhile, use a piece of gauze to wick away the urine.
   NOTE: Be careful to not contaminate the surgical field.
5. Begin disinfecting the abdomen by applying an iodine-based or chlorhexidine-based scrub and 70% alcohol with cotton-tipped swabs. Start in the center of the abdomen and work outward in a circular motion 3x. Allow the area to briefly dry between applications.
6. Check for a lack of a toe-pinch response to ensure anesthesia is adequate. Ensure the nose cone and limbs are secured. Place a surgical drape over the mouse, with the opening directly over the surgically prepped abdomen.
   NOTE: Do not drag the drape across the mouse to avoid potential contamination.

5. Surgical induction of AAA

1. Entering the abdominal cavity:
   1. Wash hands and wear clean nitrile or sterile surgical gloves. Before contacting the surgical field, always spray gloves with 70% EtOH and rub gloved hands together until dry.
   2. Use blunt forceps to tent the skin in the midline abdomen. Use surgical scissors to make a small nick in the skin, then extend the incision longitudinally, roughly 2-3 cm in length.
   3. Use forceps to lift the rectus muscles to identify the translucent linea alba. Use scissors to enter the abdominal cavity through the linea alba, then extend along the linea alba proximally and distally.
2. Exposing the abdominal aorta:
   1. Dampen a strip of gauze and two cotton-tipped swabs with warmed saline. Create an abdominal roll by tightly rolling one end of the gauze halfway, leaving a generous tail.
   2. Use a skin retractor to retract the right abdominal wall.
   3. Using dampened cotton-tipped swabs, perform a right-medial visceral rotation by gently sweeping the small and large intestines to the left upper quadrant and visualize the aorta and inferior vena cava (IVC). Use an abdominal roll to retract the bowel out of view – tuck the rolled end of the gauze underneath the bowel, and then bring the tail end around and out of the body to gently swaddle the bowel. Apply gentle tension to the tail of the gauze to hold the bowel out of the field of view. Adjust the abdominal roll and skin retractor to gain an optimal view of the retroperitoneal organs, as shown in Figure 2A.
      NOTE: The abdominal roll helps to keep the bowel moist and protect it from being inadvertently damaged by surgical instruments. Be sure the gauze remains damp during the procedure to prevent the bowel from drying out. Be careful to not forcefully retract the bowel as this can cause kinking of the superior mesenteric artery and bowel vasculature, which can potentially cause ischemic damage. Moreover, when initially sweeping the small intestine, be cautious of a thin translucent attachment between the large bowel and the inferior liver (hepatocolic ligament), which if not careful, can easily tear off the liver capsule and cause bleeding. If there is tension on this ligament during retraction, divide it sharply with scissors.

Figure 2: Representation of abdominal retraction and the optimal surgical view for exposure of the mouse infrarenal aorta. (A) Placement of a gauze abdominal roll helps to retract the intraabdominal organs, while an opposing retractor assists in providing visualization of the retroperitoneum. A sterile surgical drape (transparent to show animal orientation) is placed over the anesthetized animal to help maintain sterility. (B) The retroperitoneal fascia (green box) overlies the aorta anteriorly. (C) Example of the infrarenal aorta following dissection of the retroperitoneal fascia. Isolation of the aorta from the IVC can be achieved by starting at a potential space between the aorta and IVC located just distal to the left renal vein as it crosses anteriorly (yellow circle). Abbreviation: IVC = inferior vena cava. Please click here to view a larger version of this figure.

3. Circumferential dissection and isolation of the infrarenal aorta:
   1. Confirm that the IVC and infrarenal aorta are in full view. Begin exposing the aorta by first entering and dividing the retroperitoneal (RP) fascia (Figure 2B). Identify the gonadal (testicular or ovarian) arteries that run parallel along the anterior infrarenal aorta (Figure 2B and Figure 3). Use forceps to bluntly divide the fascia between the gonadal arteries and continue longitudinally to expose the aorta anteriorly (Figure 2C).
      NOTE: The RP fascia is a thin, translucent layer of connective tissue that contains lymphatics and the splanchnic plexus. It is necessary to dissect through the RP fascia to expose the aorta. However, do not dissect through the connective tissue of the aortic adventitia. A tear in the adventitia (white connective tissue) will expose the media (appears bright red), and the aorta will likely rupture at this site once elastase is applied.
   2. Next, start to isolate the abdominal aorta off the IVC. Begin this dissection at a small gap between the IVC and aorta, located just below the inferior edge of the left renal vein as it crosses the aorta (Figure 2C). Use the tips of forceps to gently spread apart the connective tissue fibers between the aorta and IVC and continue working circumferentially around the aorta at this level.
      NOTE: The IVC is very thin-walled and closely adheres to the aorta by a fine layer of fibrous connective tissue. Be careful to avoid handling the IVC or cleaning off the IVC as much as possible. Dissecting the right side of aorta off the IVC first (before dissecting the left side of the aorta from the surrounding musculature) will help the aorta to "fall away" from the IVC.
   3. Continue bluntly dissecting the plane between the aorta and IVC, working caudally toward the aortic bifurcation. Stop further distal dissection once reaching the aortic bifurcation.
      NOTE: Take extra precaution when dissecting around the inferior mesenteric artery (IMA), which is typically located near the mid-section of the infrarenal aorta and travels laterally across the IVC.
   4. Once the right edge of the aorta is separated from the IVC, return proximally to the level of the left renal vein. Dissect the RP fascia off the lateral left edge of the aorta, working circumferentially around until the aorta is fully isolated. See Figure 3 for relevant anatomy of the retroperitoneal dissection.
      NOTE: Be careful dissecting behind the aorta as there is high variability in the location and number of lumbar veins and arteries. See Figure 4 for reference of areas at high risk for bleeding with this dissection.
   5. Carefully inspect that the aorta is circumferentially isolated from the IVC and the surrounding musculature as much as possible, with careful dissection around the aortic segments tethered by the IMA and lumbar arteries.
   6. Place a strip of glove along the right and left edges of the aorta, as demonstrated in Figure 5A. Attempt to cover as much of the IVC as possible.
   7. Use handheld calipers to measure the widest aortic diameter and record three measurements. Spray the tips of the calipers with 70% EtOH before and after measurements. Avoid directly contacting the aorta with the caliper tips to prevent contamination.
      NOTE: Photos using a calibrated camera-capable microscope can also be utilized.

Figure 3: Anatomy of the blood supply to the lower abdomen, pelvis, and retroperitoneum of the mouse. Abbreviations: R = right; L = left. Please click here to view a larger version of this figure.

Figure 4: Sites of high risk for injury and hemorrhage during the retroperitoneal dissection and circumferential isolation of the infrarenal aorta. Abbreviations: L = left; IMA = inferior mesenteric artery. Please click here to view a larger version of this figure.

Figure 5: Intraoperative responses to elastase application or sham during the elastase/BAPN murine AAA model. (A) Segments of glove are placed along the length of the aorta prior to elastase application to help protect the IVC and bowel from exposure to elastase while keeping the aorta soaked in elastase (B) Application of denatured elastase does not cause dilation of the aorta (blue box). The maximal aortic diameter measured 0.627 mm at baseline, then 0.607 mm after 5 min of topical denatured elastase. (C) Application of elastase causes aortic dilation after 5 min of treatment. In this example, the aorta (green) dilated to 0.953 mm from 0.607 mm, a 57% increase in diameter. Abbreviations: BAPN = ß-aminopropionitrile; AAA = abdominal aortic aneurysm. Please click here to view a larger version of this figure.

4. Elastase application:
   1. Use a cotton-tipped swab to dab any extra blood or fluid off the aorta.
   2. Next, place a 10 mm x 2 mm piece of dry gauze on top of the aorta. Use a pipette to dispense 5 µL of elastase (or control denatured elastase) to saturate the gauze and aorta. Gently fold the pieces of the glove around the aorta.
      NOTE: To prepare denatured elastase for use in sham or control groups, boil elastase at 100 °C for 30 min.
   3. Allow 5 min for elastase to act on the aorta. During this incubation period, if required, release some of the tension placed by the abdominal roll and the skin retractor.
      NOTE: Due to the batch effect with elastase, we encourage investigators to use the same bottle of elastase for all experiments within a given study. With each new bottle of elastase, we recommend performing a dose-response to ensure there is not an overwhelming number of early ruptures (prior to 4 weeks). The duration of elastase application can also be adjusted between 4 and 6 min depending on the response to elastase.
   4. After 5 min, reset bowel retraction and unfold the pieces of the glove. Irrigate the abdominal cavity with 1 mL of warm 0.9% sterile normal saline, while carefully removing the gauze and pieces of glove off the aorta. Absorb the saline in the abdomen with 10 cm x 10 cm gauze. Repeat irrigating the abdomen for a total of 3 x 3 mL.
   5. Use handheld calipers to remeasure the widest aortic diameter post elastase application and record 3x. See Figure 5B,C for examples of the aortic dilation to treatment with sham and active elastase.
      NOTE: The averages of the three pre- and post- elastase measurements can be used to calculate the percent change in aortic diameter with treatment. Typically, there is a notable dilation ~30-50% immediately after elastase treatment, which can help to ensure the elastase is functional and the aorta was adequately treated. The diameter of the aorta should not change with the application of denatured elastase or may measure slightly smaller (likely from spasm).
5. Closure of the abdominal cavity:
   1. Carefully remove the abdominal roll from underneath the bowel and out of the body. If needed, apply additional saline to the bowel to prevent sticking to the abdominal roll during removal. Check to make sure the bowel appears pink and adequately perfused.
      NOTE: It is not necessary to try and reposition the bowel back into its original location; attempting so can risk twisting of the bowel or internal hernias.
   2. Reapproximate the abdominal fascia using a running 5-0 non-absorbable monofilament suture. Close the skin with 3-4 skin staples.

6. Postoperative animal care

1. Place the mouse in the recovery cage with a heat lamp. Ensure the temperature of the cage is warm, not hot.
2. Administer a 0.5-1 mL subcutaneous fluid bolus of 0.9% normal saline.
3. Allow the mouse to recover by itself in the warmed cage for ~20 min until active as per institutional protocol, then return to a housing cage.
4. Per institutional protocol, administer carprofen 20 mg/kg at 24 h after surgery on postoperative day 1, and continue daily for 3 days.

7. Aortic measurement and tissue harvest

1. Following euthanasia with isoflurane and cervical dislocation, reopen the abdominal cavity. Extend the incision through the sternum to access the thorax. Excise the right atria and perfuse the left ventricle with 10 mL of cold 1% DPBS solution over 2 min. Resect the lungs, liver, and spleen.
   NOTE: Be careful to not injure the bowel; spillage of enteric contents can impact tissue analysis.
2. Expose the abdominal aorta, and measure the maximal infrarenal aorta diameter, as described above. Continue to dissect the entire aorta and heart. Once the heart and aorta are isolated, cut all arterial branches and common iliac arteries leaving short segments intact on the aorta. Place the heart and aorta on a contrasted background next to a ruler and image.

8. Data analysis and reporting

1. To help account for human error, measure aortic diameters at least 3x each when using handheld calipers, then report the diameter as the averaged value.
2. Define AAA as a 50% increase in the healthy aorta diameter. Be sure to include both the gross aortic diameters and the percent change in diameter in the study results.