Name: biodegradable magnesium stent treatment of saccular aneurysms in a rat model - introduction of the surgical technique
Uploaded: 2017-10-01T13:00:00.000+00:00
Duration: 11 min 7 s
Description: Watch this Scientific Journal Video about Biodegradable Magnesium Stent Treatment of Saccular Aneurysms in a Rat Model - Introduction of the Surgical Technique at JoVE.com

We thank Eugen Hofmann and Philine Zumstein for their excellent technical assistance and for sharing their expertise in stent application procedures. We thank Majlinda Kalanderi for the anatomical drawing.

Male Wistar rats with a mean weight of 592 g (&#177;50 SD) and mean age of 20 weeks were housed in animal facilities at a room temperature of 22-24 &#176;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
<ol>
	<li>Use a quiet, aseptic operating room and maintain the temperature at 23 &#177; 3 &#176;C.</li>
	<li>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.</li>
	<li>Obtain the following consumable supplies to create the sidewall aneurysm and implant the stent.
	<ol>
		<li>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.</li>
		<li>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.</li>
		<li>Obtain 0.9% isotonic sodium chloride solution for irrigation/volume substitution and to ensure the cleanliness of the instruments during surgery</li>
	</ol>
	</li>
	<li>Obtain the following instruments to implant the stent: puncture needle 19G, hydrophobic guide wire, 4 Fogaty sheath, and an inflator syringe for balloon dilatation.</li>
	<li>Obtain the following consumable supplies for aneurysm wall decellularization:
	<ol>
		<li>Obtain microtubes for aneurysm storage at -24 &#176;C and labels for identification, as well as a laboratory shaker to ensure full chemical treatment of the aneurysm.</li>
		<li>Obtain 0.1% sodium dodecyl sulfate (SDS) and phosphate buffered saline (PBS) for the proper decellularization process.</li>
	</ol>
	</li>
	<li>Obtain the following standard surgery instruments:
	<ol>
		<li>Obtain coarser surgical instruments: surgical scissors, soft tissue forceps, soft tissue spreader, straight and curved forceps, three Mosquito clamps, needle holder.</li>
		<li>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.</li>
	</ol>
	</li>
</ol>
2. Anesthesia
<ol>
	<li>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.</li>
	<li>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).</li>
	<li>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.</li>
	<li>Place the rat under the infrared heating lamp until the intraperitoneal anesthesia takes effect, as preoperative warming effectively avoids extensive cooling during surgery.</li>
</ol>
3. Surgical preparation and positioning of the rat
<ol>
	<li>Weigh the rat, extract the tongue to secure the upper airways, and apply eye ointment to avoid corneal desiccation.</li>
	<li>Use adhesive tape to fix the rat to a small washable board and avoid any pressure or traction to the paws.</li>
	<li>Shave the rat in the operational area with a small animal shaver and disinfect the surgical region.</li>
	<li>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.</li>
	<li>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&#39;s hands.</li>
	<li>Wash and disinfect hands and put on sterile gloves. It is useful to have an assistant present especially for the stent implantation.</li>
	<li>Perform the surgery under aseptic conditions according to the recommendations for laboratory animals6,7.</li>
</ol>
4. Aneurysm graft harvesting
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>When the rib cage is open, sacrifice the rat with an intracardiac overdose of ketamine hydrochloride (120 mg/kg).</li>
	<li>Mobilize the left lung and fix it to the right side with the help of a cellulose swab.</li>
	<li>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.</li>
	<li>Dissect the aorta using a microsurgical technique with micro-scissors and micro-forceps.</li>
	<li>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.</li>
	<li>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.</li>
	<li>After measuring the aneurysm, immediately transplant the graft into the recipient rat.</li>
</ol>
5. Aneurysm creation and stent application
<ol>
	<li>Surgical approach
	<ol>
		<li>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.</li>
		<li>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.</li>
		<li>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.</li>
		<li>Protect the bladder and testicles from dehydration and extensive manipulation during surgery by covering them with a wet sterile cellulose swab.</li>
		<li>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.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Abdominal aorta dissection 
	NOTE: To facilitate the description of the surgical strategy we have divided the abdominal aorta into three distinctive segments (Figure 1).
	<ol>
		<li>Start the dissection in the distal segment - which is technically more challenging. 
		&#8203;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.
		<ol>
			<li>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.</li>
			<li>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.</li>
			<li>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.</li>
		</ol>
		</li>
		<li>Dissect the aorta from the V. cava in the middle segment in the same way as described before. 
		&#8203;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.
		<ol>
			<li>Identify the superior mesenteric artery at its origin from the abdominal aorta and preserve it during all procedures.</li>
			<li>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.</li>
			<li>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.</li>
			<li>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.</li>
		</ol>
		</li>
		<li>Finally, dissect the proximal segment of the abdominal aorta. This is technically simpler than the same procedure in the distal and middle segments. 
		&#8203;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.
		<ol>
			<li>After opening the parietal peritoneum, separate the vessels with two blunt forceps.</li>
			<li>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.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Aneurysm creation
	<ol>
		<li>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.</li>
		<li>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.</li>
		<li>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.</li>
		<li>Flush the artery in both directions with saline in a blunt tipped needle.</li>
		<li>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.</li>
		<li>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.</li>
		<li>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.</li>
		<li>Rinse the anastomosis site and cut the remaining ends of ligature at the aneurysm dome.</li>
		<li>Observe the distal abdominal aorta patency with the direct milking test.</li>
		<li>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.</li>
		<li>To avoid damage from prolonged ischemia, allow for 10 minutes of reperfusion before continuing with the proximal aortic clamping and stent insertion.</li>
		<li>Empty the bladder before implanting the stent.</li>
	</ol>
	</li>
	<li>Stent implantation 
	NOTE: It is useful to have an assistant on hand for the following steps, especially for the final stent inflation.
	<ol>
		<li>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.</li>
		<li>Place a large temporary clamp in the proximal segment to avoid major hemorrhaging during arterial puncture.</li>
		<li>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.</li>
		<li>Flush the artery thoroughly following the puncture with saline in both directions using a blunt tipped needle.</li>
		<li>Insert a hydrophobic guide wire into the puncture site and remove the proximal clamp in the distal segment.</li>
		<li>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.</li>
		<li>Push the dilator upwards just proximal to the anastomosis site, the sheath is located about half a centimeter below.</li>
		<li>Remove the guide wire and dilator, and ensure the 3-way stopcock is turned off to avoid unnecessary loss of blood.</li>
		<li>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).</li>
		<li>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.</li>
		<li>After adequate stent dilation and adhesion to the aortic vessel wall, deflate and remove the balloon.</li>
		<li>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.</li>
		<li>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.</li>
		<li>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.</li>
		<li>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.</li>
		<li>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.</li>
	</ol>
	</li>
</ol>
6. Closure
<ol>
	<li>Remove all gauze swabs and gently flush the abdominal cavity with isotone saline to clear any debris and promote volume substitution.</li>
	<li>Remove the soft-tissue spreader and return the small intestines, cecum, and fat mass back to their correct anatomical position.</li>
	<li>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.</li>
	<li>After closure, disinfect the skin and place the animal under the infrared light to provide it with warmth.</li>
</ol>

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C., James, B., Khachigian, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+model+of+in-stent+restenosis:+rat+aortic+stenting.">A novel model of in-stent restenosis: rat aortic stenting.</a> Heart. 91 (3), 393-395 (2005).</li><li>Finn, A. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+rat+model+of+carotid+artery+stenting+for+the+understanding+of+restenosis+in+metabolic+diseases.">A novel rat model of carotid artery stenting for the understanding of restenosis in metabolic diseases.</a> J Vasc Res. 39 (5), 414-425 (2002).</li></ol>

This work was supported by the research funds of the Research Council, Kantonsspital Aarau, Aarau, Switzerland. The biodegradable magnesium stents were supplied by Biotronik AG, Center for Vascular Intervention, Buelach, Switzerland. The authors are solely responsible for the design and conduct of the presented study and declare no competing interests.

Bioabsorbable stents and animal models 
In recent years the general trend in medicine has been away from permanent implants (which remain in the patient&#39;s body for the rest of their life) to bio-absorbable materials. Magnesium stents, in particular, are already quite established in cardiology8,9. Unfortunately these stents have not yet been tested for other applications, such as cerebrovascular diseases. For this reason we decided to stu...

Subarachnoid hemorrhage due to a ruptured intracranial aneurysm is associated with a high mortality rate and poor neurological outcome in many survivors. There are currently two general approaches to occlude IA: either microsurgical clipping (which requires operative exposure of the aneurysm), or endovascular occlusion. As the less invasive endovascular coil treatment of narrow-necked IA has been shown to be associated with slightly lower morbidity (especially in the posterior circulation1,2), endovascular treatment options have become the preferred modality of many neurosurgical centers. Numerous devices have been developed in order to extend the indications of endovascular treatment and overcome the main limitation of IA recurrence after coiling. Intracranial stents are especially promising to overcome these limitations, as they serve as a scaffold for neo-endothelization and coil herniation prevention, as well as protect the parent artery and improve intraluminal intraaneurysmal thrombosis caused by reduction of blood inflow. There is a need to study novel intracranial stents in a low cost animal model; at both macroscopic and molecular levels.
The aim of this study was to design a safe, fast, and standardized surgical technique for stent application in an already established saccular aneurysm model in rats3,4,5. In the present project, we evaluated the role of a biodegradable magnesium stent.

<table><tbody><tr><td>Medetomidine</td><td></td><td></td><td>any generic</td></tr><tr><td>Ketamin</td><td></td><td></td><td>any generic</td></tr><tr><td>Buprenorphine</td><td></td><td></td><td>any generic</td></tr><tr><td>Phosphate buffered saline</td><td></td><td></td><td></td></tr><tr><td>Sodium dodecyl sulfate (0.1%)</td><td></td><td></td><td></td></tr><tr><td>3-0 resorbable suture</td><td>Ethicon Inc., USA</td><td>VCP428G</td><td></td></tr><tr><td>5-0 non absorbable suture</td><td>Ethicon Inc., USA</td><td>8618G</td><td></td></tr><tr><td>6-0 non-absorbable suture</td><td>B. Braun, Germany</td><td>C0766070</td><td></td></tr><tr><td>9-0 non-absorbable suture</td><td>B. Braun, Germany</td><td>G1111140</td><td></td></tr><tr><td>10-0 non-absorbable suture</td><td>Covidien, USA</td><td>N2530 Monosof</td><td></td></tr><tr><td>Operation microscope</td><td>Zeiss, Germany</td><td></td><td></td></tr><tr><td>Digital microscope camera</td><td>Sony, Japan</td><td>HXR-MC1P</td><td></td></tr><tr><td>Standard surgical instruments</td><td></td><td>multiple</td><td>see protocol 7.a</td></tr><tr><td>Microsurgical instruments</td><td></td><td>multiple</td><td>see protocol 7.b</td></tr><tr><td>Vascular clip applicator</td><td>B. Braun, Germany</td><td>FT495T</td><td></td></tr><tr><td>Temporary vascular clamps</td><td>B. Braun, Germany</td><td></td><td></td></tr><tr><td>19G Puncture needle&amp;nbsp;</td><td>Angiomed GmbH, Germany</td><td>15820010</td><td></td></tr><tr><td>Hydrophobic guide wire</td><td>Cook Medical, USA</td><td>G00650</td><td></td></tr><tr><td>4F sheat</td><td>Cordis Corporation, USA</td><td>504-604A</td><td></td></tr><tr><td>Inflation syringe</td><td></td><td></td><td></td></tr><tr><td>Laboratory shaker</td><td>Stuart</td><td>SRT6</td><td></td></tr><tr><td>Magnesium Stent 2.5/6 AMS with Polymer coating</td><td>Biotronik, Switzerland</td><td></td><td></td></tr><tr><td>Surgery drape&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>Sterile cellulose swabs</td><td></td><td></td><td></td></tr><tr><td>Syringes 1 ml and 2 ml</td><td></td><td></td><td></td></tr><tr><td>Hollow needles 18G and 26G</td><td></td><td></td><td></td></tr><tr><td>Isotonic sodium chloride</td><td></td><td></td><td></td></tr><tr><td>Microtubes</td><td></td><td></td><td></td></tr><tr><td>Eye ointment</td><td>Bausch + Lomb Inc, USA</td><td>Lacrinorm</td><td>any generic</td></tr><tr><td>Small animal shaver</td><td></td><td></td><td></td></tr></tbody></table>

biodegradable magnesium stent treatment of saccular aneurysms in a rat model - introduction of the surgical technique

The steady progess in the armamentarium of techniques available for endovascular treatment of intracranial aneurysms requires affordable and reproducable experimental animal models to test novel embolization materials such as stents and flow diverters. The aim of the present project was to design a safe, fast, and standardized surgical technique for stent assisted embolization of saccular aneurysms in a rat animal model.
Saccular aneurysms were created from an arterial graft from the descending aorta.The aneurysms were microsurgically transplanted through end-to-side anastomosis to the infrarenal abdominal aorta of a syngenic male Wistar rat weighing &#62;500 g. Following aneurysm anastomosis, aneurysm embolization was performed using balloon expandable magnesium stents (2.5 mm x 6 mm). The stent system was retrograde introduced from the lower abdominal aorta using a modified Seldinger technique.
Following a pilot series of 6 animals, a total of 67 rats were operated according to established standard operating procedures. Mean surgery time, mean anastomosis time, and mean suturing time of the artery puncture site were 167 &#177; 22 min, 26 &#177; 6 min and 11 &#177; 5 min, respectively. The mortality rate was 6% (n=4). The morbidity rate was 7.5% (n=5), and in-stent thrombosis was found in 4 cases (n=2 early, n=2 late in stent thrombosis).
The results demonstrate the feasibility of standardized stent occlusion of saccular sidewall aneurysms in rats - with low rates of morbidity and mortality. This stent embolization procedure combines the opportunity to study novel concepts of stent or flow diverter based devices as well as the molecular aspects of healing.

The average duration of surgery was 167 (&#177; 22) min, 26 (&#177; 6) min of which were needed for aneurysm creation and a further 23 (&#177; 7) min required for stent application and reconstruction of the arteriotomy (Figure 3).
Mortality, morbidity, and macroscopic in-stent thrombosis were the primary endpoints of the study. The regular follow-up periods were 7 days (n=28), and 21 days (n=32) res...

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

Reproducable experimental animal models are needed for the testing of novel embolization materials, which have been designed to treat endovascular occlusion of intracranial aneurysms (IA). The present study aims to develop a safe and standardized surgical technique for stent assisted embolization of saccular aneurysms in a rat animal model.

The steady progess in the armamentarium of techniques available for endovascular treatment of intracranial aneurysms requires affordable and reproducable ...

biodegradable-magnesium-stent-treatment-saccular-aneurysms-rat-model

Research

JoVE Journal

Neuroscience

11.0K Views.  Kantonsspital Aarau. Reproducable experimentele dierlijke modellen nodig zijn voor het testen van materialen van de roman embolisatie kan leiden, die zijn ontworpen voor de behandeling van de endovasculair occlusie van intracraniële aneurysmata (IA). De huidige studie is gericht op het ontwikkelen van een veilige en gestandaardiseerde chirurgische techniek voor stent bijgestaan embolisatie kan leiden van saccular aneurysma in een diermodel van de rat.

Video: Biodegradable Magnesium Stent Treatment of Saccular Aneurysms in a Rat Model - Introduction of the Surgical Technique

A Murine Model of Stent Implantation in the Carotid Artery for the Study of Restenosis

Despite the considerable progress made in the stent development in the last decades, cardiovascular diseases remain the main cause of death in western countries. Beside the benefits offered by the development of different drug-eluting stents, the coronary revascularization bears also the life-threatening risks of in-stent thrombosis and restenosis. Research on new therapeutic strategies is impaired by the lack of appropriate methods to study stent implantation and restenosis processes. Here, we describe a rapid and accessible procedure of stent implantation in mouse carotid artery, which offers the possibility to study in a convenient way the molecular mechanisms of vessel remodeling and the effects of different drug coatings.

A model of stent implantation in mouse carotid artery is described. Compared to other similar methods, this procedure is very rapid, simple and accessible, offering the possibility to study in a convenient way the vascular wall reaction to different drug-eluting stents and the molecular mechanisms of restenosis.

Despite  the considerable progress made in the stent development in the last decades,  cardiovascular diseases remain the main cause of death in western ...

Medicine

A New Murine Model of Endovascular Aortic Aneurysm Repair

Endovascular aneurysm exclusion is a validated technique to prevent aneurysm rupture. Long-term results highlight technique limitations and new aspects of Abdominal aortic aneurysm (AAA) pathophysiology. There is no abdominal aortic aneurysm endograft exclusion model cheap and reproducible, which would allow deep investigations of AAA before and after treatment. We hereby describe how to induce, and then to exclude with a covered coronary stentgraft an abdominal aortic aneurysm in a rat. The well known elastase induced AAA model was first reported in 19901 in a rat, then described in mice2. Elastin degradation leads to dilation of the aorta with inflammatory infiltration of the abdominal wall and intra luminal thrombus, matching with human AAA. Endovascular exclusion with small covered stentgraft is then performed, excluding any interactions between circulating blood and the aneurysm thrombus. Appropriate exclusion and stentgraft patency is confirmed before euthanasia by an angiography thought the left carotid artery. Partial control of elastase diffusion makes aneurysm shape different for each animal. It is difficult to create an aneurysm, which will allow an appropriate length of aorta below the aneurysm for an easy stentgraft introduction, and with adequate proximal and distal neck to prevent endoleaks. Lots of failure can result to stentgraft introduction which sometimes lead to aorta tear with pain and troubles to stitch it, and endothelial damage with post op aorta thrombosis. Giving aspirin to rats before stentgraft implantation decreases failure rate without major hemorrhage. Clamping time activates neutrophils, endothelium and platelets, and may interfere with biological analysis.

Histological and biochemical modifications after aneurysm endograft exclusion are still unclear. We describe a new model of endograft implantation on a murine aneurysm. Through thrombus analysis with and without persistant circulating blood stream interface, and new endovascular biomaterials evaluation, this model will help to better understand endovascular aneurysm exclusion pathobiology.

Endovascular aneurysm exclusion is a validated technique to prevent aneurysm rupture. Long-term results highlight technique limitations and new aspects of ...

Imaging In-Stent Restenosis: An Inexpensive, Reliable, and Rapid Preclinical Model

Preclinical models of restenosis are essential to unravel the pathophysiological processes that lead to in-stent restenosis and to optimize existing and future drug-eluting stents.
A variety of antibodies and transgenic and knockout strains are available in rats. Consequently, a model for in-stent restenosis in the rat would be convenient for pathobiological and pathophysiological studies. 
In this video, we present the full procedure and pit-falls of a rat stent model suitable for high throughput stent research. We will show the surgical procedure of stent deployment, and the assessment of in-stent restenosis using the most elegant technique of OCT (Optical Coherence Tomography). This technique provides high accuracy in assessing plaque CSAs (cross section areas) and correlates well with histological sections, which require special and time consuming embedding and sectioning techniques. OCT imaging further allows longitudinal monitoring of the development of in-stent restenosis within the same animal compared to one-time snapshots using histology.

This video demonstrates how to use a preclinical inexpensive and reliable model to study pathobiological and pathophysiological processes of in-stent restenosis development. Longitudinal in vivo monitoring using OCT (Optical Coherence Tomography) and analysis of OCT images are also demonstrated.

Preclinical models of restenosis are essential to unravel the pathophysiological processes that lead to in-stent restenosis and to optimize existing and ...

Biology

The Helsinki Rat Microsurgical Sidewall Aneurysm Model

Experimental saccular aneurysm models are necessary for testing novel surgical and endovascular treatment options and devices before they are introduced into clinical practice. Furthermore, experimental models are needed to elucidate the complex aneurysm biology leading to rupture of saccular aneurysms.
Several different kinds of experimental models for saccular aneurysms have been established in different species. Many of them, however, require special skills, expensive equipment, or special environments, which limits their widespread use. A simple, robust, and inexpensive experimental model is needed as a standardized tool that can be used in a standardized manner in various institutions.
The microsurgical rat abdominal aortic sidewall aneurysm model combines the possibility to study both novel endovascular treatment strategies and the molecular basis of aneurysm biology in a standardized and inexpensive manner. Standardized grafts by means of shape, size, and geometry are harvested from a donor rat&#39;s descending thoracic aorta and then transplanted to a syngenic recipient rat. The aneurysms are sutured end-to-side with continuous or interrupted 9-0 nylon sutures to the infrarenal abdominal aorta.
We present step-by-step procedural instructions, information on necessary equipment, and discuss important anatomical and surgical details for successful microsurgical creation of an abdominal aortic sidewall aneurysm in the rat.

Microsurgical sidewall aneurysms in rats are created by end-to-side anastomosis of an aortic graft to the abdominal aorta. We present step-by-step instructions and discuss anatomical and surgical details for successful experimental saccular aneurysm creation.

Experimental saccular aneurysm models are necessary for testing novel surgical and endovascular treatment options and devices before they are introduced ...

Use of a Rat Model to Study Ventral Abdominal Hernia Repair

Ventral abdominal hernia is a relatively common clinical condition that sometimes requires herniorraphy (surgical repair). The repair of ventral abdominal hernia typically requires implantation of a material to serve as a mechanical bridge across the defect in the abdominal wall. Biomaterials, such as porcine small intestinal submucosa (SIS), also serve as a lattice for cell growth into the implant and can naturally incorporate into the host tissue. Development of such repair materials benefits from use of animal models in which experimental abdominal wall defects are easily created and are amenable to repair in a reproducible fashion. The method offered here describes surgical creation and repair of ventral abdominal hernia in a rat model. When SIS is used to repair an experimental ventral abdominal hernia in this model, it is rapidly incorporated into host tissue within 28 days of implantation. Histologically, incorporation of their implanted material into host tissue is characterized by a robust fibrovascular response. Future refinements and applications of the rat abdominal hernia model may likely involve diabetic and/or obese animals as a means to more closely mimic common co-morbidities of man.

This manuscript describes methods associated with creation and repair of a ventral abdominal wall defect (hernia). This model can be used to study repair strategies such as those that use implanted materials. In this manuscript, repair of the experimental hernia with porcine small intestinal submucosa is presented as an example.

Ventral abdominal hernia is a relatively common clinical condition that sometimes requires herniorraphy (surgical repair). The repair of ventral abdominal ...

Bioengineering

Vascular Gene Transfer from Metallic Stent Surfaces Using Adenoviral Vectors Tethered through Hydrolysable Cross-linkers

In-stent restenosis presents a major complication of stent-based revascularization procedures widely used to re-establish blood flow through critically narrowed segments of coronary and peripheral arteries. Endovascular stents capable of tunable release of genes with anti-restenotic activity may present an alternative strategy to presently used drug-eluting stents. In order to attain clinical translation, gene-eluting stents must exhibit predictable kinetics of stent-immobilized gene vector release and site-specific transduction of vasculature, while avoiding an excessive inflammatory response typically associated with the polymer coatings used for physical entrapment of the vector. This paper describes a detailed methodology for coatless tethering of adenoviral gene vectors to stents based on a reversible binding of the adenoviral particles to polyallylamine bisphosphonate (PABT)-modified stainless steel surface via hydrolysable cross-linkers (HC). A family of bifunctional (amine- and thiol-reactive) HC with an average t1/2 of the in-chain ester hydrolysis ranging between 5 and 50 days were used to link the vector with the stent. The vector immobilization procedure is typically carried out within 9 hr&#160;and consists of several steps: 1) incubation of the metal samples in an aqueous solution of PABT (4 hr); 2) deprotection of thiol groups installed in PABT with tris(2-carboxyethyl) phosphine (20 min); 3) expansion of thiol reactive capacity of the metal surface by reacting the samples with polyethyleneimine derivatized with pyridyldithio (PDT) groups (2 hr); 4) conversion of PDT groups to thiols with dithiothreitol (10 min); 5) modification of adenoviruses with HC (1 hr); 6) purification of modified adenoviral particles by size-exclusion column chromatography (15 min) and 7) immobilization of thiol-reactive adenoviral particles on the thiolated steel surface (1 hr). This technique has wide potential applicability beyond stents, by facilitating surface engineering of bioprosthetic devices to enhance their biocompatibility through the substrate-mediated gene delivery to the cells interfacing the implanted foreign material.

These studies report on reversible attachment of adenoviral gene vectors to coatless metal surfaces of stents and model mesh disks. Sustained release of transduction-competent viral particles contingent upon hydrolysis of cross-linkers used for vector immobilization results in a durable site-specific transgene expression in vascular cells and in stented arteries.

In-stent restenosis presents a major complication of stent-based revascularization procedures widely used to re-establish blood flow through critically ...

Creation of a Rodent Model of Abdominal Aortic Aneurysm by Blocking Adventitial Vasa Vasorum Perfusion

The adventitial vasa vasorum (VV) provides oxygen and nourishment to the aortic wall. Hypoxia in the aortic wall can cause enlarged abdominal aortic aneurysms (AAAs). This article introduces and describes a standard protocol used to induce AAAs through adventitial VV hypoperfusion created with a combination of polyurethane catheter insertion into the aortic lumen and suture ligation of the infrarenal abdominal aorta.
The protocol involves the use of male rats weighing 300-400 g, which are provided food and water ad libitum. After laparotomy with a ventral midline abdominal incision, exfoliation of the aorta is performed, which blocks blood flow from the perivascular tissue. Aortotomy involving a small incision adjacent to the renal artery branches is performed, and a polyurethane catheter is inserted using an 18-gauge indwelling needle. After repairing the incision, tight ligation of the aorta over the catheter blocks VV blood flow from the proximal direction through the aortic wall without disturbing the aortic blood flow. This technique can induce an AAA with progressive aortic dilatation.
The greatest benefit of this model is that VV hypoperfusion causes tissue hypoxia and the development of an infrarenal AAA, which has morphological and pathological characteristics similar to those of a human AAA.

Polyurethane catheter insertion into the aortic lumen and suture ligation of the aorta induce chronic hypoxia due to hypoperfusion of the adventitial vasa vasorum. This article describes a novel animal model of abdominal aortic aneurysm (AAA) with characteristics similar to those of AAA in humans.

The adventitial vasa vasorum (VV) provides oxygen and nourishment to the aortic wall. Hypoxia in the aortic wall can cause enlarged abdominal aortic ...

A Rat Model of Orthotopic Liver Transplantation Using a Novel Magnetic Anastomosis Technique for Suprahepatic Vena Cava Reconstruction

The rat model of orthotopic liver transplantation (OLT) is essential for transplant research. It is a very sophisticated animal model and requires a steep learning curve. The introduction of the cuff technique for anastomosis of the portal vein (PV) and infrahepatic vena cava (IHVC) has significantly simplified the transplant procedure in rats. However, due to the short anterior wall of the recipients' suprahepatic vena cava (SHVC), the cuff technique is very difficult to use for the reconstruction of the SHVC. Most researchers in this field still use the hand-suture technique for SHVC reconstruction, which makes it the bottleneck step in rat orthotopic liver transplantation. The magnetic anastomosis technique (i.e., magnamosis) is a method of connecting two vessels using the attractive force between two magnets. Our recent study has shown that the magnetic anastomosis technique is superior to the hand-suture technique for SHVC reconstruction in rats. In this article, we show a step-by-step protocol for SHVC reconstruction in rats using the novel magnetic anastomosis technique. In this model, the reconstruction of the PV and IHVC was performed by the standard cuff technique, while the reconstruction of the bile duct (BD) was performed by a stent technique. The hepatic re-arterialization was not performed. The magnetic anastomosis technique made SHVC reconstruction much easier and significantly shortened the anphepatic phase. After a reasonable learning curve, even researchers without advanced microsurgical skills can produce reliable and reproducible results using this rat model of OLT.

The reconstruction of the suprahepatic vena cava (SHVC) remains a difficult step in rat orthotopic liver transplantation. In this article, we show a step-by-step protocol for SHVC reconstruction in rats using a novel magnetic anastomosis technique.

The rat model of orthotopic liver transplantation (OLT) is essential for transplant research. It is a very sophisticated animal model and requires a steep ...

Implantation of Human-Sized Coronary Stents into Rat Abdominal Aorta Using a Trans-Femoral Access

Percutaneous coronary intervention (PCI), combined with the deployment of a coronary stent, represents the gold standard in interventional treatment of coronary artery disease. In-stent restenosis (ISR) is determined by an excessive proliferation of neointimal tissue within the stent and limits the long-term success of stents. A variety of animal models have been used to elucidate pathophysiological processes underlying in-stent restenosis (ISR), with the porcine coronary and the rabbit iliac artery models being the most frequently used. Murine models provide the advantages of high throughput, ease of handling and housing, reproducibility, and a broad availability of molecular markers. The apolipoprotein E deficient (apoE-/- ) mouse model has been widely used to study cardiovascular diseases. However, stents must be miniaturized to be implanted into mice, involving important changes of their mechanical and (potentially) biological properties. The use of apoE-/- rats can overcome these shortcomings as apoE-/- rats allow for the evaluation of human-sized coronary stents while at the same time providing an atherogenic phenotype. This makes them an excellent and reliable model to investigate ISR after stent implantation. Here, we describe, in detail, the implantation of commercially available human coronary stents into the abdominal aorta of rats with an apoE-/- background using a trans-femoral access.

This protocol describes the implantation of human coronary stents into the abdominal aorta of rats with an apoE-/- background using a trans-femoral access. Compared with other animal models, murine models carry the advantages of high throughput, reproducibility, ease of handling and housing, and a broad availability of molecular markers.

Percutaneous coronary intervention (PCI), combined with the deployment of a coronary stent, represents the gold standard in interventional treatment of ...

Using a Cell-Tracer Injection to Investigate the Origin of Neointima-Forming Cells in a Rat Saccular Side Wall Model

Microsurgical clipping creates a subsequent barrier of blood flow into intracranial aneurysms, whereas endovascular treatment relies on neointima and thrombus formation. The source of endothelial cells covering the endoluminal layer of the neointima remains unclear. Therefore, the aim of the present study was to investigate the origin of neointima-forming cells after cell-tracer injection in the already well-established Helsinki rat microsurgical sidewall aneurysm model.
Sidewall aneurysms were created by suturing decellularized or vital arterial pouches end-to-side to the aorta in male Lewis rats. Before arteriotomy with aneurysm suture, a cell-tracer injection containing CM-Dil dye was performed into the clamped aorta to label endothelial cells in the adjacent vessel and track their proliferation during follow-up (FU). Treatment followed by coiling (n = 16) or stenting (n = 15). At FU (7 days or 21 days), all rats underwent fluorescence angiography, followed by aneurysm harvesting and macroscopic and histological evaluation with immunohistological cell counts for specific regions of interest. 
None of the 31 aneurysms had ruptured upon follow-up. Four animals died prematurely. Macroscopically residual perfusion was observed in 75.0% coiled and 7.0% of stented rats. The amount of cell-tracer-positive cells was significantly elevated in decellularized stented compared to coiled aneurysms with respect to thrombus on day 7 (p = 0.01) and neointima on day 21 (p = 0.04). No significant differences were found in thrombus or neointima in vital aneurysms. 
These findings confirm worse healing patterns in coiled compared to stented aneurysms. Neointima formation seems particularly dependent on the parent artery in decellularized aneurysms, whereas it is supported by the recruitment from aneurysm wall cells in vital cell-rich walls. In terms of translation, stent treatment might be more appropriate for highly degenerated aneurysms, whereas coiling alone might be adequate for aneurysms with mostly healthy vessel walls.

We performed a one-point, lipophilic cell-tracer injection to track endothelial cells, followed by an arteriotomy and suturing of sidewall aneurysms on the abdominal rat aorta. Neointima formation seemed dependent on the parent artery in decellularized aneurysms and was promoted by the recruitment from aneurysm wall cells in vital cell-rich walls.

Microsurgical clipping creates a subsequent barrier of blood flow into intracranial aneurysms, whereas endovascular treatment relies on neointima and ...

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

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