The authors are solely responsible for the design and conduct of the presented study. Dr. Marbacher was supported by a grant from the Swiss National Science Foundation (PBSKP3-123454). The authors declare no conflict of interests. 
Author contributions to the study and manuscript preparation include the following. Conception and design: SM, JM, JF. Acquisition of data: SM, EA, JF. Analysis and interpretation of data: SM, JF, JM. Drafting the article: SM, JF, JM. Critically revising the article: JH, MN. Statistical analysis: SM, JF. Study supervision: JF, JH, MN.

NOTE: Male Wistar rats (mean body weight: 356 &#177; 44 g; 10-14 weeks old) were housed in the animal room at 22-24 &#176;C and twelve hour light/dark cycle with free access to pellet diet, regular tap water and also received humane care in conformity with institutional guidelines. The experiments were reviewed and approved by the Committee for Animal Welfare at the University of Helsinki, Finland. &#160;
NOTE: In the following demonstration our surgical method is as follows: Anesthetize the rat by weight-adapted subcutaneous injection of medetomidine hydrochloride (0.5 mg/kg) and intraperitoneal injection of ketamine hydrochloride (50 mg/kg). Test for the lack of a toe-pinch reflex to confirm that the rat is fully anesthetized. Apply eye ointment, clip the surgical site, and clean the skin with a suitable disinfectant, for example Chlorhexidine, either in alcohol or water. Wash hands, put on protective clothing, a head cover and facemask, and sterile surgical gloves. Have a surgical assistant aid in maintaining aseptic surgery conditions and to document the surgical characteristics (as listed in Table 1). Monitor the depth of the anesthesia every 15 min during surgery by following respiratory rate, heart rate, and reaction to noxious stimulation (toe pinch test). Subcutaneous injection of buprenorphine (0.03 mg/kg) was given for postoperative analgesia and repeated if necessary every 12 hr.
1. Hardware, Consumables, and Positioning
<ol>
	<li>Keep the small animal surgery room quiet, aseptic, and maintain the room temperature at 23 +/- 3 &#176;C. To perform the experimental aneurysm surgery the following minimal equipment is necessary:
	<ol>
		<li>Use a table top surgical microscope ideally equipped with an assistant scope and digital microscope camera. Use nonporous reusable operating surface and cleanable instrument surface to protect the laboratory bench.</li>
		<li>Prepare consumables such as skin disinfectants, physiological saline, smaller and larger gauze swabs and suture material including 9-0 micro-sutures, a 5-0 non-absorbable, and 3-0 absorbable suture.</li>
		<li>Use the following standard surgical instruments: Surgical scissors, tissue forceps, soft tissue spreader or self-retaining retractor, and two Mosquito surgical clamps.</li>
		<li>Use a basic microsurgical instrument set that includes: Curved microneedle holder, one curved and two straight microforceps, and a straight or curved microscissor.</li>
		<li>Keep the microsurgical instruments in a kidney dish filled with sterile saline to keep the instrument non-sticky and clean during surgery. The kidney dish is padded with rubber mat or a surgical glove to prevent damage to the tips of the micro-instruments. Ensure that all supplies are sterile and the procedure is performed with aseptic technique according to the current recommendations for survival surgery on laboratory animals8-10.</li>
		<li>In addition, use a vascular clip applicator and three atraumatic temporary vascular clamps. It is important that the clamps used have a low closing power to prevent injury to the very thin wall of the rat aorta. Also prepare a ruler with half millimeter scale bars, a small colored rubber pad, and a short blunt needle.</li>
		<li>Place the rats in a supine position, immobilize both front and hind paws with surgical tape without applying stretch or compression to the skin, and bend their back with a thick marker or cautery pen by placing it under the lumber region of the back. It is important to obtain as much lumbar spine lordosis as possible in order to improve retroperitoneal exposure and access to the infrarenal aorta which facilitates microsurgical anastomosis. This positioning is recommended for abdominal aorta aneurysm creation but not necessary for thoracic graft harvesting.</li>
	</ol>
	</li>
</ol>
2. Graft Harvesting
<ol>
	<li>Under general anesthesia, open the thoracic cavity (start graft ischemia time). Apply a noxious toe pinch to confirm that the rat is unresponsive before proceeding with the following steps. Cut through the midventral abdominal wall, identify the diaphragm just above the liver, and cut the connective tissue at the bottom of diaphragm to allow access to the rib cage. With large scissors, blunt side down, cut through ribs just one centimeter left and right of the rib cage midline and open up the thoracic cavity. The lungs are mobilized to the right side of the heart. Sacrifice the rats by overdosing with intracardiac injection of ketamine hydrochloride.Pulmonary trunk, left subclavian vein, left cranial vena cava, and the azygous vein interfere with the harvest of the proximal thoracic aorta.</li>
	<li>Caudal to the prominent veins there is a good entry point to start the dissection of the descending thoracic aorta using micro-scissors and microforceps.</li>
	<li>Trace the thoracic aorta back from the dorsal wall of the thorax upwards to the aortic arch by gentle blunt retraction and dissection with the Mosquito surgical clamp.</li>
	<li>Clamp and then cut the veins with scissors. Maintain the clamps on the veins and use as a retractor to expose the underlying aortic arch.</li>
	<li>Place a non-absorbable 6-0 silk ligature just above the first intercostal artery leaving the aorta.</li>
	<li>Cut the descending aorta just below the left subclavian artery and then below the ligature. Trimming can be done in order to get a perpendicular standardized aneurysm geometry, or if needed, to get a specific angle between the axis of the aneurysm and the aorta. Measure the graft in its width and length. 
	NOTE: Harvested grafts can either be immediately transplanted into recipient rats or further processed to achieve decellularization of the graft wall. Decellularized grafts can be stored at -4 degree Celsius until re-implantation at a later date (Figure 1). The decellularization of the aneurysm wall has been shown to predispose the aneurysm to enlarge11.</li>
</ol>
3. Graft Decellularization
<ol style="margin-left: 40px;">
	<li>Freeze donor grafts in phosphate buffered saline at -4 &#176;C.</li>
	<li>The next day, thaw the grafts, rinse with purified and deionized water at room temperature, and incubate for 10 hr at 37 &#176;C in 0.1% sodium dodecyl sulfate.</li>
	<li>Finally, wash the sodium dodecyl sulfate-treated grafts three times with gentle agitation, refreeze in phosphate buffered saline, and keep at -4 &#176;C until use.</li>
</ol>
4. Aneurysm Creation
<ol style="margin-left: 40px;">
	<li>Dissection of the Abdominal Aorta
	<ol>
		<li>After the animal has been anaesthetized, clip the fur from the surgical site and clean the skin with a suitable disinfectant. Test for the lack of a toe-pinch response prior to skin incision. Start the incision 1cm proximal to the genitals in midventral position (start operation time). Carefully separate the skin from the underlying muscles. End dissection one to two centimeters below the sternum.</li>
		<li>Carefully but firmly pull up the underlying abdominal muscles to avoid damage to the underlying organs. Extend the longitudinal cut along the linea alba upwards to the xiphoid process and end in caudal direction at the level of the bladder.</li>
		<li>Gently apply pressure to the bladder to empty it in order to facilitate access to the retroperitoneal space.</li>
		<li>Move the small intestines and the prominent cecum to the right or the left. Identify the large intestine namely the descending colon on the left bottom of the abdominal cavity.</li>
		<li>Cut the ligament in between the small intestine and the descending colon in cranial direction to allow wider exposure of the dorsal body wall. Place a self-retaining retractor to hold the bowels apart.</li>
		<li>The ideal location of end-to-side aneurysm anastomosis is found at the level between the renal and iliolumbar veins. The abdominal aorta lies retroperitoneal embedded in fat tissue. During dissection pay particular attention to the paired almost transparent ureters and testicular vessels.</li>
		<li>If further retraction of intestines is needed, use larger gauze swaps. The lumbar spine lordosis induced during positioning by placing a cautery pen or similar object under the lower back of the rat, reduces significantly the need of bowel retraction.</li>
		<li>The ventral surface of the dorsal body wall is covered with a thin parietal peritoneum. Once this is opened, visualize the aorta just underneath. During careful sharp and blunt dissection of the abdominal aorta from adjacent large veins grasp only the adventitia to avoid damage to the vessel wall.</li>
		<li>Infrequently, small lumbar arteries arise as segmental vessel from the dorsal surface of the abdominal aorta and interfere with preparation. Ligation and cutting of the vessel is needed to avoid retrograde oozing during aneurysm suturing. Use of curved micro-forceps can facilitate ligature placement in the depth.</li>
	</ol>
	</li>
	<li>End-to-side Anastomosis
	<ol>
		<li>Put a colored rubber pad underneath the abdominal aorta and upholster it with a small gauze swab. Remove loose connective tissue and adventitia at the level of the planned anastomosis site.</li>
		<li>Clamp the abdominal aorta distal to the anastomosis first, then proximal (start aortic clamping time). This ensures a firm filling of the vessel and facilitates subsequent arteriotomy.</li>
		<li>Perform the arteriotomy using either straight or curved micro-scissors. A micro-forceps holds up a very small piece of the vessel wall to cut out an elliptical shape.</li>
		<li>Flush the artery thoroughly with saline in both directions using a blunt tipped needle.</li>
		<li>Place the first two sutures of this end-to-side anastomosis at the proximal and distal end of the arteriotomy (start anastomosis time). Avoid grasping of the vessel wall with the micro-forceps whenever possible. Make sure that each suture is placed through all layers of the vessel wall.</li>
		<li>Perform suturing either as continuous or interrupted sutures. If interrupted suturing is chosen then place the back side nine o`clock suture first. Subsequent sutures can be spaced apart starting adjacent to the very first suture. Grasp the adventitia carefully. Avoid any squeezing/grasping of the intima.</li>
		<li>When the back wall is finished, check the endoluminal part of the anastomosis for misplaced sutures. Perform the same procedures in the same order on the front side. Ensure that the first of a total of three knots per suture is firm but not too tight.</li>
	</ol>
	</li>
	<li>Hemostasis and Closure
	<ol>
		<li>After the end to side anastomosis is completed (end anastomosis time), rinse the site with saline and remove the distal clamp first to allow for backflow (end aortic clamping time and end graft ischemia time).</li>
		<li>If obvious bleeding occurs from backflow an extra stitch may be needed (start time of hemostasis). In case of minor oozing, achieve hemostasis with gentle pressure over the bleeding site using a small piece of a gauze swab.</li>
		<li>Remove the proximal vascular clamp, rinse the anastomosis site once more, and cut off the remaining ends of the ligature at the aneurysm dome.</li>
		<li>Confirm patency of the aneurysm by observation of volume increase of the aneurysm during peak arterial pulse wave. Assess distal abdominal artery patency through the direct &#8220;milking test&#8221;.</li>
		<li>Remove the plastic sheet and the small gauze swab underneath. The pulsating blood whirl within the created aneurysm is clearly visible.</li>
		<li>Cover suture lines around the anastomosis with small pieces of adipose tissue or Spongostan for additional hemostasis if small oozing is still present (end time of hemostasis).</li>
		<li>Remove the soft-tissue spreader and gauze swabs. Place the small intestines, the cecum, and the fat mass back in their correct position.</li>
		<li>Close the midline abdominal muscles using 5-0, 4-0, or 3-0 resorbable or non-absorbable sutures followed by skin wound closure using continuous suture technique with 3-0 resorbable polyfilament suture (end operation time). Note: In our experience rats tolerated resorbable skin sutures better than non-absorbable monofilament sutures.</li>
	</ol>
	</li>
</ol>

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AJNR.</a> American journal of neuroradiology. 32, 772-777 (2011).</li><li>Frosen, J., 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=Contribution+of+mural+and+bone+marrow-derived+neointimal+cells+to+thrombus+organization+and+wall+remodeling+in+a+microsurgical+murine+saccular+aneurysm+model.">Contribution of mural and bone marrow-derived neointimal cells to thrombus organization and wall remodeling in a microsurgical murine saccular aneurysm model.</a> Neurosurgery. 58, 936-944 (2006).</li><li>Marjamaa, J., 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=Mice+with+a+deletion+in+the+first+intron+of+the+Col1a1+gene+develop+dissection+and+rupture+of+aorta+in+the+absence+of+aneurysms:+high-resolution+magnetic+resonance+imaging.">Mice with a deletion in the first intron of the Col1a1 gene develop dissection and rupture of aorta in the absence of aneurysms: high-resolution magnetic resonance imaging.</a> at 4.7 T, of the aorta and cerebral arteries. 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R., Luo, Y., Mulder, G. B., White, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Principles+of+rodent+surgery+for+the+new.">Principles of rodent surgery for the new.</a> , (2011).</li><li>Marbacher, S., 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=Loss+of+mural+cells+leads+to+wall+degeneration,+aneurysm+growth,+and+eventual+rupture+in+a+rat+aneurysm+model.">Loss of mural cells leads to wall degeneration, aneurysm growth, and eventual rupture in a rat aneurysm model.</a> Stroke. 45, 248-254 (2014).</li><li>Frosen, J., 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=Remodeling+of+saccular+cerebral+artery+aneurysm+wall+is+associated+with+rupture:+histological+analysis+of+24+unruptured+and+42+ruptured+cases.">Remodeling of saccular cerebral artery aneurysm wall is associated with rupture: histological analysis of 24 unruptured and 42 ruptured cases.</a> Stroke. 35, 2287-2293 (2004).</li><li>Frosen, J., 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=Saccular+intracranial+aneurysm:+pathology+and+mechanisms.">Saccular intracranial aneurysm: pathology and mechanisms.</a> Acta neuropathologica. , 123-773 (2012).</li><li>Ysuda, R., Strother, C. M., Aagaard-Kienitz, B., Pulfer, K., Consigny, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+large+and+giant+bifurcation+aneurysm+model+in+canines:+proof+of+feasibility.">A large and giant bifurcation aneurysm model in canines: proof of feasibility.</a> AJNR Am J Neuroradiol. 33, 507-512 (2012).</li><li>Sherif, C., 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=Microsurgical+venous+pouch+arterial-bifurcation+aneurysms+in+the+rabbit+model:+technical+aspects.">Microsurgical venous pouch arterial-bifurcation aneurysms in the rabbit model: technical aspects.</a> Journal of visualized experiments : JoVE. , (2011).</li><li>Marjamaa, J., 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=High-resolution+TOF+MR+angiography+at+4.7+Tesla+for+volumetric+and+morphologic+evaluation+of+coiled+aneurysm+neck+remnants+in+a+rat+model.">High-resolution TOF MR angiography at 4.7 Tesla for volumetric and morphologic evaluation of coiled aneurysm neck remnants in a rat model.</a> Acta Radiol. 52, 340-348 (2011).</li></ol>

The authors have no financial or commercial interest in any of the drugs, materials, or equipment used.

Progress in our understanding of the complex biology of saccular cerebral artery aneurysm depends on analysis of epidemiological and clinical data, complemented by laboratory studies on patient samples and experimental work in animal models3,12,13.
Small animals such as the rat are inherently associated with lower costs of experiments and housing, and reduced need of specialized equipment. An average total operation time of less than 60 minutes for microsurgical creation of a sidewa...

Rupture of a saccular cerebral artery aneurysm causes life threatening hemorrhage leading to stroke, permanent neurological damage, or death. Rupture can be prevented by either microsurgical clipping or endovascular aneurysm occlusion. A medical treatment to prevent aneurysm growth and rupture has not yet been established.
Experimental models for saccular aneurysms are needed to study the biology of arterial aneurysms and for the testing of novel therapeutic devices and strategies. For these purposes, several different models in different species have been developed and published1. Larger aneurysm models in pigs, dogs, and rabbits are preferably used to test endovascular innovations in complex aneurysm architecture1,2. Murine aneurysm models, on the other hand, allow testing research questions in genetically modified species3,4 and facilitate clarification of aneurysm biology at cellular and molecular level far better than larger species1. Although endovascular trans-carotid and trans-iliac device deployment is limited to bigger rats (&#62;400-500 g) and stents smaller than 2.0 mm and 1.5 mm in diameter5,6, stents can also be placed through direct insertion into the abdominal aortic segment harbouring the experimental aneurysms. Previous work using the rat microsurgical abdominal aortic sidewall aneurysm model demonstrated its feasibility in testing novel embolic devices and its use in studying the molecular basis of aneurysm biology3,7.
Many of the currently published experimental saccular aneurysm models require expensive equipment, special environments (e.g. sterile operation rooms with fluoroscopy capabilities), interventional radiology competence, or use of expensive species. These requirements limit the widespread use of these models, and lead to the use of different models in different laboratories, which makes data comparison and meta-analysis difficult, if not impossible. A simple, robust, and inexpensive experimental model is needed as a standardized tool that can be used in a standardized manner in various labs in order to get comparable results from different institutions. For this purpose, we created the rat aorta sidewall saccular arterial aneurysm model.
The aim of this report is to present step-by-step procedural instructions, information on necessary equipment, and to discuss important anatomical and surgical characteristics for successful microsurgical creation of abdominal aortic sidewall aneurysms in the rat.

<table><tbody><tr><td>Medetomidine</td><td></td><td></td><td>Any genericon</td></tr><tr><td>Ketamin</td><td></td><td></td><td>Any genericon</td></tr><tr><td>Buprenorphine</td><td></td><td></td><td>Any genericon</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>&lt;a name=&quot;RANGE!C7&quot;&gt;VCP824G&lt;/a&gt;</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 silk suture</td><td>B. Braun, Germany</td><td>C0761060</td><td></td></tr><tr><td>9-0 nylon micro suture</td><td>B. Braun, Germany</td><td>G1118471</td><td></td></tr><tr><td>Spongostan</td><td>Ethicon Inc., USA</td><td>MS0002</td><td></td></tr><tr><td>Operation microscope</td><td>Leica , Germany</td><td>M651</td><td></td></tr><tr><td>Digital microscope camera</td><td>Sony, Japan</td><td>SSC-DC58AP</td><td></td></tr><tr><td>Standard surgical instruments</td><td>B. Braun, Germany</td><td>Multiple</td><td>See protocol 1.4</td></tr><tr><td>Microsurgical instruments</td><td>B. Braun, Germany</td><td>Multiple</td><td>See protocol 1.5</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>FT250T</td><td></td></tr><tr><td>Graph Pad Prism statistical software&amp;nbsp;</td><td>GraphPad Software, San Diego, California, USA</td><td></td><td>V 6.02 for Windows</td></tr></tbody></table>

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.

A pilot series comprised 14 rats. Subsequently a total of 84 animals were operated according to the presented protocol for several research projects between March and September 2012. Additional 29 animals served as donors for arterial saccular grafts. The remaining experiments were performed using pre-treated grafts harvested and stored from previous experiments using rats of the same gender, strain, weight, and age.
Body weight, overall operation time, aortic clamping time, time for anastomos...

Watch this Scientific Journal Video about The Helsinki Rat Microsurgical Sidewall Aneurysm Model at JoVE.com

The Helsinki Rat Microsurgical Sidewall Aneurysm Model

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 ...

the-helsinki-rat-microsurgical-sidewall-aneurysm-model

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JoVE Journal

Medicine

12.2K Views.  University of Helsinki, Helsinki, Finland. 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.

Video: The Helsinki Rat Microsurgical Sidewall Aneurysm Model

Microsurgical Venous Pouch Arterial-Bifurcation Aneurysms in the Rabbit Model: Technical Aspects

For ruptured human cerebral aneurysms endovascular embolization has become an equivalent alternative to aneurysm clipping.1 However, large clinical trials have shown disappointing long-term results with unacceptable high rates of aneurysm recanalization and delayed aneurysm rupture.2 To overcome these problems, animal experimental studies are crucial for the development of better endovascular devices.3-5
Several animal models in rats, rabbits, canines and swine are available.6-8 Comparisons of the different animal models showed the superiority of the rabbit model with regard to hemodynamics and comparability of the coagulation system and cost-effectiveness.9-11 
The venous pouch arterial bifurcation model in rabbits is formed by a venous pouch sutured into an artificially created true bifurcation of both common carotid arteries (CCA). The main advantage of this model are true bifurcational hemodynamics.12 The major drawbacks are the sofar high microsurgical technical demands and high morbidity and mortality rates of up to 50%.13 These limitations have resulted in less frequent use of this aneurysm model in the recent years. These shortcomings could be overcome with improved surgical procedures and modified peri- and postoperative analgetic management and anticoagulation.14-16 Our techniques reported in this paper demonstrate this optimized technique for microsurgical creation of arterial bifurcation aneurysms.

An optimized technique for the microsurgical creation of arterial bifurcation aneurysms mimicking bifurcation human cerebral aneurysms is described. A venous pouch is sutured into an artificially created true bifurcation of both common carotid arteries. Facilitated microsurgical techniques and aggressive postoperative anticoagulation and analgesia lead to minimized morbidity rates and high aneurysm patency rates.

For ruptured human cerebral aneurysms endovascular embolization has become an equivalent alternative to aneurysm clipping.1 However, large clinical trials ...

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 ...

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.

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 ...

Neuroscience

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 ...

Arterial Pouch Microsurgical Bifurcation Aneurysm Model in the Rabbit

Endovascular treatment for intracranial aneurysms gained importance over the past decades, consequently there is an increased need of testing endovascular devices. Animal models respecting rheological, hemodynamic and aneurysm wall conditions are highly warranted. Therefore, the aim of the present study was to design a novel standardized and reproducible surgical technique to create autologous arterial pouch bifurcation aneurysms with non-modified and modified wall conditions in rabbits.
Bifurcation aneurysms were created by end-to-side anastomosis of the right on the left common carotid artery, both serving as parent arteries for the arterial pouch, which was microsurgically sewn on. Grafts were taken from the proximal right common carotid artery, either for the control (n = 7, immediate autologous re-implantation) or modified (n = 7, incubated with 100 international units elastase for 20 minutes before autologous re-implantation) group. Pouch and parent artery patency were controlled by fluorescence angiography immediately after creation. At follow-up (28 days), all rabbits underwent contrast enhanced magnetic resonance angiography and fluorescence angiography followed by aneurysm harvesting, macroscopic and histological evaluation.
A total of 16 female New Zealand White rabbits were operated upon. Two animals died prematurely. At follow-up, 85.72% of all aneurysms remained patent. Both groups revealed an increase in aneurysm size over time; this was more pronounced in the control group (6.48 &#177; 1.81 mm3 at time of creation vs. 19.85 &#177; 6.40 mm3 at follow-up, p = 0.037) than in the modified group (8.03 &#177; 1.08 mm3 at time of creation vs. 20.29 &#177; 6.16 mm3 at follow-up, p = 0.054).
Our findings demonstrate the adequacy of this new rabbit model which allows for the creation of bifurcation aneurysms with different wall conditions in a microsurgical approach. Given the excellent long-term patency and the property of aneurysm growth over time, this model may serve as an important tool for preclinical evaluation of novel endovascular therapies.

Developing and testing endovascular devices for intracranial aneurysm treatment is still of great importance. Most aneurysm models used today miss either the important characteristics of an arterial degenerated wall or the hemodynamics of a true bifurcation. Therefore, we aimed to design a novel arterial pouch bifurcation model in rabbits.

Endovascular treatment for intracranial aneurysms gained importance over the past decades, consequently there is an increased need of testing endovascular ...

Murine Surgical Model of Topical Elastase Induced Descending Thoracic Aortic Aneurysm

According to the Center for Disease Control, aortic aneurysms (AAs) were considered a leading cause of death in all races and both sexes from 1999-2016. An aneurysm forms as a result of progressive weakening and eventual dilation of the aorta, which can rupture or tear once it reaches a critical diameter. Aneurysms of the descending aorta in the chest, called descending thoracic aortic aneurysms (dTAA), make up a large proportion of aneurysm cases in the United States. Uncontained dTAA rupture is almost universally lethal, and elective repair has a high rate of morbidity and mortality. The purpose of our model is to study dTAA specifically, to elucidate the pathophysiology of dTAA and to search for molecular targets to halt the growth or reduce the size of dTAA. By having a murine model to study thoracic pathology precisely, targeted therapies can be developed to specifically test dTAA. The method is based on the placement of porcine pancreatic elastase (PPE) directly on the outer murine aortic wall after surgical exposure. This creates a destructive and inflammatory reaction, which weakens the aortic wall and allows for aneurysm formation over weeks to months. Though murine models possess limitations, our dTAA model produces robust aneurysms of predictable size. Furthermore, this model can be used to test genetic and pharmaceutical targets which may arrest dTAA growth or prevent rupture. In human patients, interventions such as these could help avoid aneurysm rupture, and difficult surgical intervention.

We describe a surgical protocol to consistently induce robust descending thoracic aortic aneurysms in mice. The procedure involves left thoracotomy, thoracic aorta exposure, and placement of a sponge soaked in porcine pancreatic elastase on the aortic wall.

According to the Center for Disease Control, aortic aneurysms (AAs) were considered a leading cause of death in all races and both sexes from 1999-2016. ...

Endovascular Perforation Model for Subarachnoid Hemorrhage Combined with Magnetic Resonance Imaging (MRI)

The endovascular filament perforation model to mimic subarachnoid hemorrhage (SAH) is a commonly used model - however, the technique can cause a high mortality rate as well as an uncontrollable volume of SAH and other intracranial complications such as stroke or intracranial hemorrhage. In this protocol, a standardized SAH mouse model is presented, induced by endovascular filament perforation, combined with magnetic resonance imaging (MRI) 24 h after operation to ensure the correct bleeding site and exclude other relevant intracranial pathologies. Briefly, C57BL/6J mice are anesthetized with an intraperitoneal ketamine/xylazine (70 mg/16 mg/kg body weight) injection and placed in a supine position. After midline neck incision, the common carotid artery (CCA) and carotid bifurcation are exposed, and a 5-0 non-absorbable monofilament polypropylene suture is inserted in a retrograde fashion into the external carotid artery (ECA) and advanced into the common carotid artery. Then, the filament is invaginated into the internal carotid artery (ICA) and pushed forward to perforate the anterior cerebral artery (ACA). After recovery from surgery, mice undergo a 7.0 T MRI 24 h later. The volume of bleeding can be quantified and graded via postoperative MRI, enabling a robust experimental SAH group with the option to perform further subgroup analyses based on blood quantity.

Endovascular Perforation Model for Subarachnoid Hemorrhage Combined with Magnetic Resonance Imaging MRI

Here we present a standardized SAH mouse model, induced by endovascular filament perforation, combined with magnetic resonance imaging (MRI) 24 h after operation to ensure the correct bleeding site and exclude other relevant intracranial pathologies.

The endovascular filament perforation model to mimic subarachnoid hemorrhage (SAH) is a commonly used model - however, the technique can cause a high ...

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 ...

Creation of Two Saccular Elastase-Digested Aneurysms with Different Hemodynamics in One Rabbit

Preclinical animal models with hemodynamic, morphologic, and histologic characteristics close to human intracranial aneurysms play a key role in the understanding of the pathophysiological processes and the development and testing of new therapeutic strategies. This study aims to describe a new rabbit aneurysm model that allows the creation of two elastase-digested saccular aneurysms with different hemodynamic conditions within the same animal.
Five female New Zealand white rabbits with a mean weight of 4.0 (&#177; 0.3) kg and mean age of 25 (&#177;5) weeks underwent microsurgical stump and bifurcation aneurysm creation. One aneurysm (stump) was created by right common carotid artery (CCA) exposure at its origin at the brachiocephalic trunk. A temporary clip was applied at the CCA origin and another, 2 cm above. This segment was treated with a local injection of 100 U of elastase for 20 min. A second aneurysm (bifurcation) was created by suturing an elastase-treated arterial pouch into the end-to-side anastomosis of the right CCA to left CCA. Patency was controlled by fluorescence angiography immediately after creation.
The average duration of surgery was 221 min. The creation of two aneurysms in the same animal was successful in all rabbits without complication. All aneurysms were patent immediately after surgery except for one bifurcation aneurysm, which showed an extreme tissue reaction due to elastase incubation and an immediate intraluminal thrombosis. No mortality was observed during surgery and up to one-month follow-up. Morbidity was limited to a transient vestibular syndrome (one rabbit), which recovered spontaneously within one day.
Demonstrated here for the first time is the feasibility of creating a two-aneurysm rabbit model with stump and bifurcation hemodynamic characteristics and highly degenerated wall conditions. This model allows the study of the natural course and potential treatment strategies on the basis of aneurysm biology under different flow conditions.

This protocol describes the steps for the creation of a rabbit model with two elastase-digested aneurysms with different hemodynamics (stump and bifurcation constellation). This allows the testing of novel endovascular devices in aneurysms with different angioarchitecture and hemodynamic conditions within a single animal.

Preclinical animal models with hemodynamic, morphologic, and histologic characteristics close to human intracranial aneurysms play a key role in the ...

Microsurgical Creation of Giant Bifurcation Aneurysms in Rabbits for the Evaluation of Endovascular Devices

Giant aneurysms are dangerous lesions requiring endovascular treatment, with high rates of aneurysm recanalization and re-rupture. Reliable in vivo models are rare but are required for testing new endovascular devices.&#160;We demonstrate the technical aspects of the creation of giant bifurcation aneurysms in New Zealand white rabbits (2.5-5.5 kg). A 25-30 mm long venous pouch is taken from the external jugular vein, and a bifurcation between both carotid arteries is created microsurgically. The pouch is sutured in the bifurcation to mimic a giant aneurysm. This protocol summarizes our previously published standard technique for venous pouch true arterial bifurcation aneurysms and highlights its essential modification steps for giant aneurysms. Using this modified technique, we were able to create an animal model for giant aneurysms with high comparability to humans regarding the hemodynamics and coagulation systems. Furthermore, low morbidity and high aneurysm patency rates were achieved. The proposed giant aneurysm model offers an excellent possibility for testing new endovascular devices.

Here, we describe the technique for the microsurgical creation of giant bifurcation aneurysms in rabbits for the evaluation of endovascular devices.

Giant aneurysms are dangerous lesions requiring endovascular treatment, with high rates of aneurysm recanalization and re-rupture. Reliable in vivo models ...