Biodegradable Magnesium Stent Treatment of Saccular Aneurysms in a Rat Model – Introduction of the Surgical Technique

Male Wistar rats with a mean weight of 592 g (±50 SD) and mean age of 20 weeks were housed in animal facilities at a room temperature of 22-24 °C and twelve hour light/dark cycle, with free access to tap water and a pellet diet. The animals received care from humans in accordance with institutional guidelines. The experiments were approved by the Committee for Animal Care of the Canton Bern, Switzerland (BE 102/13). We strictly followed the recommendations for Animal Research: Reporting of In Vivo Experiments (ARRIVE guidelines).

1. Laboratory equipment, consumable supplies, surgical instruments

1. Use a quiet, aseptic operating room and maintain the temperature at 23 ± 3 °C.
2. Use a surgical desk with a surface which is easy to clean and disinfect, and cover it with a surgery drape. Use a table-top surgical microscope with a camera to record the surgery and an infrared light to warm the animal before and after surgery.
3. Obtain the following consumable supplies to create the sidewall aneurysm and implant the stent.
   1. Obtain smaller and larger sterile cellulose and gauze swabs, syringes with 18G and 26G hollow needles and a blunt tipped needle for irrigation/volume substitution.
   2. Obtain suture materials including non-absorbable 10-0 and 9-0 micro suture, non-absorbable 6-0 and 5-0 suture, and absorbable 3-0 suture.
   3. Obtain 0.9% isotonic sodium chloride solution for irrigation/volume substitution and to ensure the cleanliness of the instruments during surgery
4. Obtain the following instruments to implant the stent: puncture needle 19G, hydrophobic guide wire, 4 Fogaty sheath, and an inflator syringe for balloon dilatation.
5. Obtain the following consumable supplies for aneurysm wall decellularization:
   1. Obtain microtubes for aneurysm storage at -24 °C and labels for identification, as well as a laboratory shaker to ensure full chemical treatment of the aneurysm.
   2. Obtain 0.1% sodium dodecyl sulfate (SDS) and phosphate buffered saline (PBS) for the proper decellularization process.
6. Obtain the following standard surgery instruments:
   1. Obtain coarser surgical instruments: surgical scissors, soft tissue forceps, soft tissue spreader, straight and curved forceps, three Mosquito clamps, needle holder.
   2. Obtain surgical micro instruments: straight and curved micro-scissors, straight and curved micro-forceps, and four temporary vascular mini clips with corresponding vascular clip applicators.

2. Anesthesia

1. Place the rat in a gas chamber and anesthetize with a mixture of isoflurane and oxygen (4% isoflurane). Maintain inhalation until the animal loses consciousness.
2. Remove the anesthetized animal from the gas chamber and inject the definitive anesthesia consisting of a weight-adapted mixture of medetomidine hydrochloride (0.5 mg/kg) and ketamine hydrochloride (50 mg/kg) intraperitoneally (by injecting into the right or left lower abdomen).
3. Monitor the depth of anesthesia in defined intervals during surgery with a noxious toe-pinch to confirm that the rat is unresponsive and fully anesthetized. If there is a toe-pinch reflex, readminster the anesthesia with a weight-adapted intraperitoneal injection of ketamine and medetomidine. Sufficient anesthesia is required before opening the abdominal cavity, and performing the end-to-side anastomosis and stent insertion.
4. Place the rat under the infrared heating lamp until the intraperitoneal anesthesia takes effect, as preoperative warming effectively avoids extensive cooling during surgery.

3. Surgical preparation and positioning of the rat

1. Weigh the rat, extract the tongue to secure the upper airways, and apply eye ointment to avoid corneal desiccation.
2. Use adhesive tape to fix the rat to a small washable board and avoid any pressure or traction to the paws.
3. Shave the rat in the operational area with a small animal shaver and disinfect the surgical region.
4. Place two thick markers 2-3 cm apart under the back of the animal to cause as much lumbar and thoraco-lumbar lordosis as possible. This ensures better exposition of the retroperitoneum and easier application of the stent.
5. Cover the rat with a sterile aperture drape and place it under the operating microscope. Set up two supports beside the animal to facilitate a more comfortable position for the experimenter's hands.
6. Wash and disinfect hands and put on sterile gloves. It is useful to have an assistant present especially for the stent implantation.
7. Perform the surgery under aseptic conditions according to the recommendations for laboratory animals^6,7.

4. Aneurysm graft harvesting

1. Under general anesthesia, open the abdomen below the xiphoid process. First, cut the skin in the midventral line with sharp scissors and separate the skin from the musculature. Then cut the abdominal musculature and identify the diaphragm above the liver at the insertion point of the thoracic cavity.
2. Cut the diaphragm and the connective tissue with sharp scissors to access the thoracic cavity and cut the ribs with large scissors one and a half centimeter to the right and left of the sternum.
3. When the rib cage is open, sacrifice the rat with an intracardiac overdose of ketamine hydrochloride (120 mg/kg).
4. Mobilize the left lung and fix it to the right side with the help of a cellulose swab.
5. Following appropriate exposure of the aortic arc, clamp the azygos and left cranial caval veins with two mosquito clamps above the aorta and cut in between. Leave the clamps on the cut vessel endings to avoid retrograde venous bleeding. Use the clamps to move the heart and veins to the side, allowing better access to the aortic arc and descending aorta.
6. Dissect the aorta using a microsurgical technique with micro-scissors and micro-forceps.
7. Trace the aorta upwards from the descending section to the aortic arch and identify the left subclavian artery and the first intercostal artery leaving the aorta.
8. Place a non-absorbable 6-0 suture just cranial of the first intercostal artery and tie it up. At the proximal end, cut the aorta right after the origin of the left subclavian artery. It is important to use a single perpendicular cut as it lowers the chance of irregularities at the cutting edge of the vascular graft. Finally, remove the newly created aneurysm with a cut just below the ligature.
9. After measuring the aneurysm, immediately transplant the graft into the recipient rat.

5. Aneurysm creation and stent application

1. Surgical approach
   1. Start the skin incision in the distal third of the abdomen with sharp scissors and cut in a midventral position up to 1 cm distal of the xiphoid process. Carefully separate the skin and subcutaneous tissue from the musculature. Use the same scissors to open the abdominal muscles along the linea alba. Use forceps to raise the muscles while cutting to avoid damaging the intraabdominal organs.
   2. Following the laparotomy, move the small intestines, the caecum, and the colon to the right. Use a self-holding retractor to keep the abdominal cavity open and fix the organs. Place a swab beneath the liver to remove it from the surgical area and enhance retroperitoneal exposure.
      NOTE: If the fat mass is obstructing the view, it can be placed outside the abdominal cavity and covered with wet gauze to avoid extensive fluid depletion.
   3. Empty the urinary bladder to achieve maximal exposure of the infrarenal abdominal aorta. This can be accomplished either through gentle pressure to the bladder wall, or vesical discharge using a 26G hollow needle.
   4. Protect the bladder and testicles from dehydration and extensive manipulation during surgery by covering them with a wet sterile cellulose swab.
   5. Open the parietal peritoneum covering the aorta with two blunt forceps. Take care with the almost transparent ureters, testicular vessels and the superior mesenteric artery. As soon as the parietal peritoneum is open, the abdominal aorta will be located just underneath it. It is often covered by a thin layer of adipose tissue.
   6. Start with a blunt dissection of the abdominal aorta using two micro-forceps to release it from the surrounding retroperitoneal fat until a longer length of the vessel is visible. Continue with sharp dissection using micro-scissors and micro-forceps. Only grasp the adventitia during this procedure to avoid damage to the vessel wall.
2. Abdominal aorta dissection
   NOTE: To facilitate the description of the surgical strategy we have divided the abdominal aorta into three distinctive segments (Figure 1).
   1. Start the dissection in the distal segment – which is technically more challenging.
      ​NOTE: The distal segment is subdivided between the iliac bifurcation and iliolumbar vein and artery. This segment serves as the point for the distal abdominal aorta puncture and stent insertion.
      1. Dissect the aorta with micro-scissors and micro-forceps from the V. cava by cutting alongside the arterial adventitial layer between the two vessels.
         NOTE: Small lumbar arteries may arise infrequently as segmental vessels from the dorsal surface of the abdominal aorta and interfere with the procedure. Coagulation and cutting of the vessels is needed to avoid retrograde oozing during vessel suturing.
      2. Identify the right iliolumbar artery; its origin can vary significantly in this segment but is most frequently located more caudal than the left counterpart.
         NOTE: This condition rarely interferes with the planned stent insertion puncture site in the artery, but may require a puncture point farther removed or – for the most extreme anatomical variability – a vessel ligation of the right iliolumbar artery.
      3. After preparing the aorta in this segment, place a small colored rubber pad underneath the vessel to indicate where the arterial puncture will be performed. Avoid stretching the vessel in this segment. Tensile stretch on the vessel wall may lead to uncontrolled vessel rupture following arterial puncture, which in turn will impede the final reconstruction. It is therefore important to release the aorta from all adhesions at the artery puncture site and avoid any placement of gauze swabs beneath the aorta.
   2. Dissect the aorta from the V. cava in the middle segment in the same way as described before.
      ​Note: The middle segment is subdivided between the iliolumbar vein and artery and renal vein. This segment is used for the aneurysm implantation and stent positioning.
      1. Identify the superior mesenteric artery at its origin from the abdominal aorta and preserve it during all procedures.
      2. Ligate or coagulate arteries from the dorsal surface of the aorta in this section as well. To avoid damage to the lumbosacral plexus, ensure sufficient flushing and be mindful of the short coagulation time.
      3. Establish the location of the stent following dissection of the aorta in this segment. Anticipate possible interferences of the stent with abdominal vessels before defining the final location for the aneurysm and stent.
      4. After establishing the location of the aneurysm and stent, place a small piece of gauze and a colored rubber pad under the aorta for better exposure.
   3. Finally, dissect the proximal segment of the abdominal aorta. This is technically simpler than the same procedure in the distal and middle segments.
      ​Note: The proximal segment is located proximal to the renal vein. It is used for the application of the most proximal temporary clip during the Seldinger technique stent implantation.
      1. After opening the parietal peritoneum, separate the vessels with two blunt forceps.
      2. Following dorsal circumnavigation of the aorta with curved, blunt forceps, place a small piece of a colored rubber pad underneath it to facilitate later proximal clip application.
3. Aneurysm creation
   1. Clamp the aorta in the middle segment by first applying the distal clamp, followed by the proximal to ensure a firm filling of the vessel and facilitates the subsequent arteriotomy.
   2. Remove the adventitia at the planned anastomosis site. Grasp the adventitia with micro-forceps and carefully cut with micro scissors without injuring the vessel wall.
   3. Lift a small piece of vessel wall with micro-forceps and perform a linear incision with micro-scissors. The arteriotomy should be the same size as the aneurysm base.
   4. Flush the artery in both directions with saline in a blunt tipped needle.
   5. Place the first two stitches of the end-to-side anastomosis, which is performed with non-absorbable 9-0 suture, at the proximal and distal end of the arteriotomy. Grasp the vessel wall as little as possible during suturing and ensure that the sutures pass through each layer of the vessel wall. They should only catch the ventral part of the aorta to avoid constriction of the artery; which could lead to vessel rupture during stent application.
   6. Close the incision with interrupted sutures. Start the suture on the left side and finish this before continuing onto the other side. When the back wall is finished, check the endoluminal section of the anastomosis for wrongly placed sutures.
      NOTE: At this point, different intra-aneurysmal therapeutics such as coil application may be introduced.
   7. After finishing the anastomosis, remove the distal clamp first. Place an extra suture in case of major bleeding from backflow. Manage minor oozing by gently pressing a small piece of gauze to the source of bleeding. Adipose tissue or a previously obtained adventitial layer can be useful as well. Now remove the proximal clamp.
   8. Rinse the anastomosis site and cut the remaining ends of ligature at the aneurysm dome.
   9. Observe the distal abdominal aorta patency with the direct milking test.
   10. Remove the small gauze swab underneath the colored rubber pad, as all segments of the aorta should be approximately at the same height before stent insertion. Avoid covering the anastomosis suture lines with adipose tissue as the stent struts must be visible while positioning the stent beneath the newly created aneurysm.
   11. To avoid damage from prolonged ischemia, allow for 10 minutes of reperfusion before continuing with the proximal aortic clamping and stent insertion.
   12. Empty the bladder before implanting the stent.
4. Stent implantation
   NOTE: It is useful to have an assistant on hand for the following steps, especially for the final stent inflation.
   1. Clip the artery puncture site in the distal segment first. Begin with the distal clamp and follow with the proximal. Remove the loose connective tissue and (partially) the adventitia at the puncture site with micro-scissors.
   2. Place a large temporary clamp in the proximal segment to avoid major hemorrhaging during arterial puncture.
   3. Perform the arterial puncture in the distal segment with a 18G puncture needle. Lift a small piece of the vessel wall with micro-forceps to facilitate the puncture and reduce the risk of piercing both aortic vessel walls.
   4. Flush the artery thoroughly following the puncture with saline in both directions using a blunt tipped needle.
   5. Insert a hydrophobic guide wire into the puncture site and remove the proximal clamp in the distal segment.
   6. Push the guide wire forward up to the level of the clamp in the proximal segment. Insert the Teflon introducer according to the Seldinger technique using the previously inserted guide wire.
      NOTE: Hydrophobic guide wires are more suitable than hydrophilic ones as they have a superior grip and provide better manual control. As Teflon introducers are easier to insert than polypropylene introducers, they reduce the risk of uncontrolled rupture at the puncture site.
   7. Push the dilator upwards just proximal to the anastomosis site, the sheath is located about half a centimeter below.
   8. Remove the guide wire and dilator, and ensure the 3-way stopcock is turned off to avoid unnecessary loss of blood.
   9. After vacuuming the balloon system, insert the absorbable magnesium stent using the 4 Fogarty introducer. Inflate the balloon with one bar of pressure just prior to removing the introducer device to protect the endothelium of the collapsed vessel from strut-induced damage and reduce the risk of the strut becoming entangled in the previously implanted intraaneurysmal material (Figure 2).
   10. When the stent is in its final position, carefully inflate the balloon with 8-9 bar. Continuously check the end-to-side anastomosis sutures for possible ripping.
   11. After adequate stent dilation and adhesion to the aortic vessel wall, deflate and remove the balloon.
   12. When removing the introducer, have a temporary clip at hand for immediate application proximal to the puncture site as retrograde oozing from proximal patent arteries may be significant.
   13. Remove the temporary clamp in the proximal segment after placing the proximal clip in the distal segment. Observe the end-to-side anastomosis as any increase in intra-aortic blood pressure due to distal clipping may cause bleeding at the suture site.
   14. Flush the puncture site with saline from a blunt tipped needle in both directions. Then suture it with an interrupted technique using non-absorbable 10-0 thread. If possible, avoid grasping the vessel wall with the micro-forceps and ensure that each suture passes through all layers of the vessel wall. Suture at the outmost vessel puncture site to avoid vessel stenosis.
   15. Add extra sutures if major hemorrhaging occurs as a result of backflow. In case of minor oozing, secure hemostasis through gentle pressure to the bleeding site using a gauze swab or a previously preserved segment of adventitia. Small pieces of adipose tissue can be helpful as well.
   16. Remove the colored rubber pad underneath the puncture site. The pulsation of the aorta will be clearly visible proximal and distal to the anastomosis site. Check for distal artery patency with the direct milking test.

6. Closure

1. Remove all gauze swabs and gently flush the abdominal cavity with isotone saline to clear any debris and promote volume substitution.
2. Remove the soft-tissue spreader and return the small intestines, cecum, and fat mass back to their correct anatomical position.
3. Use non-absorbable 5-0 suture to join the abdominal muscles. To reduce skin traction, place some subcutaneous sutures using an absorbable 3-0 suture. Use the same 3-0 suture to close the skin.
4. After closure, disinfect the skin and place the animal under the infrared light to provide it with warmth.