The authors thank Prof Hans Rudolf Widmer, Dr Luca Remonda, and Prof Javier Fandino for their scientific support and technical contribution to this work. A special thanks to Olgica Beslac for her advice during the procedures and Kay Nettelbeck for his assistance. Furthermore, we thank Daniela Casoni DVM, PhD and med. vet. Luisana Garcia, PD Dr Alessandra Bergadano, and Dr Carlotta Detotto for their dedicated veterinary support.

NOTE: The experiment was approved by the Local Committee for Animal Care of the Canton Bern, Switzerland (Application Number BE108/16), and all animal care and procedures were performed in accordance with institutional guidelines and 3R principles15,16. Data are reported according the ARRIVE guidelines. Peri-operative management was conducted by a board-certified veterinarian anesthesiologist. For the study, female New Zealand white rabbits, with a mean weight of 4.0 (&#177; 0.3) kg and mean age of 25 (&#177;5) weeks, were housed at a room temperature of 22-24 &#176;C with a 12-h light/dark cycle with free access to water, pellets, and hay.
1. Pre-surgical phase and anesthesia
<ol>
	<li>Perform a clinical examination as recommended by the Association of veterinary Anesthetists and the European and American College of Veterinary Anesthesia and analgesia to confirm that the rabbits are healthy by weighing each animal, evaluating the mucous membrane, documenting the capillary refill time and pulse quality, and performing a pulmonary and cardiac auscultation as well as an abdominal palpation.</li>
	<li>Based on the clinical finding, attribute an American Society of Anesthesiologists (ASA) classification to each rabbit17. Perform surgery only on animals with an ASA I score.</li>
	<li>Shave both outer ears, and apply prilocaine-lidocaine cream on auricular arteries and veins. Achieve deep sedation with a combination of ketamine 20 mg/kg, dexmedetomidine 0.1&#160;mg/kg, and methadone 0.3 mg/kg injected subcutaneously (SC). Leave the animals undisturbed for 15 min. Give supplementary oxygenation (3 L/min) through a loosened face mask, and monitor with a pulse oximeter.</li>
	<li>Place a 22 G cannula in the left auricular central artery as well as in an auricular vein. Induce general anesthesia with propofol 1-2 mg/kg intravenous (IV) until effect (loss of swallowing reflex). Proceed with endo-tracheal intubation via a silicone tube (3 mm internal diameter).</li>
	<li>Shave the forehead to place the pediatric electroencephalographic (EEG) sensors. Shave the surgical field, and inject ropivacaine hydrochloride 0.75 % intradermally.</li>
	<li>Place the rabbit on the operation table in dorsal recumbency, install full monitoring, and connect the endo-tracheal tube to a low-resistance pediatric circle system. Maintain anesthesia with administration of isoflurane in oxygen, targeting a maximal end tidal (Et) concentration of 1.3 %.</li>
	<li>Provide a continuous infusion of ringer lactate 5 mL/kg/h through the venous access. Ensure clinical and instrumental monitoring until extubation by means of pulse oximetry, doppler and invasive blood pressure, 3-lead electrocardiogram, EEG, rectal temperature, and inhaled and exhaled gases.</li>
	<li>Disinfect the surgical field with povidone iodine from the manubrium sterni to jaw angles, and apply the sterile draping. During surgery, provide analgesia with lidocaine (constant rate infusion (CRI) of 50 &#956;g/kg/min) and fentanyl (CRI of 3-10 &#956;g/kg/h). Perform spontaneous or assisted ventilation. Allow permissive hypercapnia.</li>
	<li>Perform at least one arterial blood gas analysis during surgery. In case of hypotension (mean arterial pressure below 60 mmHg), treat it with noradrenaline, titrated until effect. Use a heating pad or a heating forced-air warming system to prevent hypothermia (aim: rectal temperature 37.5-38.5 &#176;C). 
	&#8203;NOTE: As the invasive arterial blood pressure is measured at the left ear artery, the clipping of the left CCA will stop the blood flow and suppress the curve. The blood pressure has then to be measured with Doppler technique until reopening of the vessel.</li>
</ol>
2. Surgery
<ol>
	<li>Approach
	<ol>
		<li>Make a median skin incision from the hyoid bone until a point 1.5 cm caudal to the manubrium sterni with a scalpel. Prepare the subcutaneous and fat tissue from the medial incision while performing meticulous hemostasis.</li>
		<li>Free the sternocephalicus muscle from the adherent connective tissue, and apply lidocaine topically (2-4 mg/kg, prefer lidocaine 1%) to avoid myoclonus. Expose the right CCA medially of the sternocephalicus muscle and keep it wet with wet swabs.</li>
		<li>Now prepare the lateral and proximal parts of the sternocephalicus muscle and retract it medially with a vessel loop to expose the CCA. Identify the external jugular vein and protect it with a wet micro swab.</li>
		<li>Dissect the connective tissue carefully along the proximal CCA until the bifurcation of the brachiocephalic trunk to expose the artery. In the presence of small branches coming from the artery, coagulate them with the cauterizer. 
		NOTE: Take care to avoid any nerve damage.</li>
	</ol>
	</li>
	<li>Stump aneurysm creation and tissue harvesting for the bifurcation aneurysm
	<ol>
		<li>Before clipping the right CCA, measure the anti-clotting time (ACT), and give natrium heparin (80 EI/kg) systemically via the ear vein (performed by the anesthesia team) to avoid thromboembolic events.</li>
		<li>Now apply 2 temporary clips: the first one at the origin of the CCA and the second one 2 cm distal from it (Figure 1A). Place a rubber pad under the vessel and rinse with papaverine HCL (40 mg/ mL; 1:1 dissolved in 0.9% saline) for vasodilatation.</li>
		<li>Remove the adventitia carefully using microscissors. Perform an arteriotomy below the distal clip with a 22 G IV-catheter, and insert the catheter caudally up to the proximal clip (Figure 1A,B).</li>
		<li>Flush the segment intraluminally with heparinized NaCl (500 U/100 mL in 0.9% saline) until there is no blood visible, and finally fix the catheter with a ligature (4-0). Now, through the catheter, inject 0.1-0.2 mL of elastase (100 IU previously dissolved in 5 mL of Tris-Buffer) into the artery segment and incubate for 20 min (Figure 1B).</li>
		<li>Start with the dissection on the left side to expose the left CCA (see section 2.3). After 20 min of incubation time with elastase, clear the elastase solution, and change the syringe to rinse the artery segment about 10 times with 0.9% NaCl.</li>
		<li>Apply 2 ligatures (6-0): the first one 5 mm distal of proximal clip and the second just proximally, under the arteriotomy (Figure 1C). Cut the vessel ~3 mm above the first ligature and one more time between the second ligature and the distal clip. Keep this autologous graft in a heparinized solution (500 U/100 mL in 0.9% saline) until the creation of the bifurcation aneurysm (Figure 1D). Finally, carefully open the first proximal clip, and measure the aneurysm (length, width, and depth).</li>
	</ol>
	</li>
	<li>Bifurcation aneurysm creation
	<ol>
		<li>Prepare the left side by dissecting the sternocephalicus muscle medially to expose ~2 cm of the left CCA. Apply lidocaine topically on the muscle to avoid myoclonus.</li>
		<li>Underlay the carotid artery with a gauze ball and a small swab with a piece of glove. Apply some papaverine.HCl topically (40 mg/mL; 1:1 dissolved in 0.9% saline). Continue to work under microscopic view: prepare the aneurysm pouch and remove the adventitia. Measure the aneurysm pouch (length, width, depth).</li>
		<li>Flush the open part of the right CCA with heparinized NaCl and if needed, replace the clip to have ~1 cm to allow free manipulations for the suture. Remove the adventitia carefully, and make a ~2 mm longitudinal incision laterally in the stump of the right CCA.</li>
		<li>Now apply two temporary clips on the left CCA to delimit a segment of ~1 cm and remove the adventitia in between. Perform an arteriotomy with a 23 G needle. Flush the segment with heparinized NaCl (500 U/100 mL in 0.9% saline). Enlarge the arteriotomy using microscissors to ~4-5 mm to allow the suturing of the right CCA and the aneurysm pouch (Figure 1E). Irrigate the vessels during the whole suturing procedure and protect them with wet micro swaps.</li>
		<li>Perform the anastomosis with 9-0 non resorbable suture.
		<ol>
			<li>Suture the proximal back wall of the right carotid blunt with 5 stitches, starting at the proximal edge of the arteriotomy on the left CCA. Then, suture the backside of the aneurysm pouch with 4-5 stitches, starting at the distal edge of the arteriotomy on the left CCA.</li>
			<li>Continue with the distal backside at the level of the fish mouth incision to suture with the vertical backside of the aneurysm graft with 3 stitches. Suture the front side of the fish mouth incision with 3 stitches, starting upwards and moving downwards.</li>
			<li>Finish with the front suture between the left CCA and front side of the aneurysm graft and right CCA with ~6 stitches. Before finishing the anastomosis, rinse the vessels with heparinized 0.9% saline solution intraluminally.</li>
		</ol>
		</li>
		<li>Before removing the clamp, measure the anti-clotting time (ACT) one more time, and administer an adapted dose of heparin systemically (target: 2-3 times baseline ACT).</li>
		<li>Remove the clip on the right CCA while putting some pressure on the anastomosis with micro swabs for hemostasis. Then, continue by removing the distal clip from the left CCA. If there is no major bleeding, continue with taking out the proximal clip on the left CCA, to allow blood flow. If there is some bleeding from the anastomose, apply some pressure with the gauze ball and swab; wait for a couple of minutes. If it persists, replace the clips and perform re-stitches. 
		NOTE: A blood loss of more than 20-30 mL can endanger the recovery phase.</li>
	</ol>
	</li>
	<li>Patency control and documentation
	<ol>
		<li>After opening all the vessels, document the results photographically and measure them (Figure 1F and Figure 2A,B).</li>
		<li>Confirm the restoration of the flow in the distal CCA through the invasive arterial blood pressure curve (measured at the ear artery, a direct branch of the external carotid), which should also return to normal.</li>
		<li>Perform fluorescence angiography by administering 1 mL of fluorescein IV, using 2 bandpass filters, a video camera, and a bicycle spotlight. See previous publications for the description of the whole procedure18,19.</li>
	</ol>
	</li>
	<li>Closure
	<ol>
		<li>Readapt the fat pad on the anastomosis and suture it with a 4-0 resorbable suture. Finally suture subcutis and skin with single stitches using 4-0 resorbable suture.</li>
	</ol>
	</li>
</ol>
3. Postsurgical phase
<ol>
	<li>At the end of the surgery, discontinue isoflurane and systemic analgesia without reversion in order to maintain the analgesic effect. Ensure that control of the swallowing reflex has returned before performing tracheal extubation.</li>
	<li>Administer meloxicam 0.5 mg/kg IV to ensure analgesia, aspirin (ASS) 10 mg/kg IV to prevent immediate thrombotic events, vitamin B12 100 &#956;g SC and clamoxyl 20 mg/kg IV as antibiotic prophylaxis.</li>
	<li>Provide supplementary oxygenation and warming until the rabbit spontaneously regains the sternal recumbency. Perform rescue analgesia with methadone if any sign of pain is observed. Perform postoperative follow-up and care 4 times a day for the first 3 preoperative days, in accordance with the guidelines for the assessment and management of pain in rodents and rabbits23,24.</li>
	<li>Ensure postoperative analgesia with fentanyl patch (12 &#956;g/h) applied on the outer ear, meloxicam 1x/ SC for 3 days, and methadone as rescue therapy, along with a score sheet for pain evaluation (Supplementary File).</li>
</ol>

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E., 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=Testing+bioresorbable+stent+feasibility+in+a+rat+aneurysm+model.">Testing bioresorbable stent feasibility in a rat aneurysm model.</a> Journal of Neurointerventional Surgery. 11 (10), 1050-1054 (2019).</li><li>Nevzati, E., 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=Biodegradable+magnesium+stent+treatment+of+saccular+aneurysms+in+a+rat+model+-+Introduction+of+the+surgical+technique.">Biodegradable magnesium stent treatment of saccular aneurysms in a rat model - Introduction of the surgical technique.</a> Journal of Visualized Experiments: JoVE. (128), e56359 (2017).</li><li>Gruter, B. E., 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=Patterns+of+neointima+formation+after+coil+or+stent+treatment+in+a+rat+saccular+sidewall+aneurysm+model.">Patterns of neointima formation after coil or stent treatment in a rat saccular sidewall aneurysm model.</a> Stroke. 52 (3), 1043-1052 (2021).</li><li>Wanderer, 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=Aspirin+treatment+prevents+inflammation+in+experimental+bifurcation+aneurysms+in+New+Zealand+White+rabbits.">Aspirin treatment prevents inflammation in experimental bifurcation aneurysms in New Zealand White rabbits.</a> Journal of NeuroInterventional Surgery. , (2021).</li><li>Lyu, Y., 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=An+effective+and+simple+way+to+establish+eastase-induced+middle+carotid+artery+fusiform+aneurysms+in+rabbits.">An effective and simple way to establish eastase-induced middle carotid artery fusiform aneurysms in rabbits.</a> Biomed Research International. 2020 (10), 1-12 (2020).</li><li>Wang, 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=Rabbit+aneurysm+models+mimic+histologic+wall+types+identified+in+human+intracranial+aneurysms.">Rabbit aneurysm models mimic histologic wall types identified in human intracranial aneurysms.</a> Journal of NeuroInterventional Surgery. 10 (4), 411-415 (2018).</li><li>Kang, W., 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+modified+technique+improved+histology+similarity+to+human+intracranial+aneurysm+in+rabbit+aneurysm+model.">A modified technique improved histology similarity to human intracranial aneurysm in rabbit aneurysm model.</a> Neuroradiology Journal. 23 (5), 616-621 (2010).</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 (10), 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=Growth+factor+receptor+expression+and+remodeling+of+saccular+cerebral+artery+aneurysm+walls:+implications+for+biological+therapy+preventing+rupture.">Growth factor receptor expression and remodeling of saccular cerebral artery aneurysm walls: implications for biological therapy preventing rupture.</a> Neurosurgery. 58 (3), 534-541 (2006).</li></ol>

The authors declare no conflict of interest.

The most common technique for aneurysm creation involves the creation of a stump aneurysm at the origin of the right CCA, either through an open or an endovascular method. The model has been validated to be a stable non-growing aneurysm that remains open with time20,21. The second possible technique involves the microsurgical creation of an arterial bifurcation aneurysm by anastomosing the right CCA on the left one and suturing an aneurysm pouch on the bifurcatio...

Intracranial aneurysm is a severe condition with a mortality rate after rupture reaching 50% and long-term disability in 10-20% of patients1. The last decade has seen a rapid development of endovascular treatment options but, at the same time, also an increasing rate of recurrence with up to 33% of aneurysm recanalization after coiling2,3. To better understand the pathophysiology underlying aneurysm occlusion and recanalization, as well as for the development and testing of new endovascular devices, there is currently a need for reliable preclinical models whose hemodynamic, morphological, and histologic characteristics mimic those of human intracranial aneurysms4,5,6. As of today, there is no defined model as a standard for preclinical trial, and a large range of species and techniques are available to researchers7,8.
However, the rabbit is a species of particular interest due to the size and hemodynamic similarities between its neck arteries and the human cerebral vessels, as well as its similar coagulation and thrombolysis profiles. Several models with elastase-digested saccular aneurysms on the common carotid arteries (CCAs) have shown qualitative and quantitative similarities with human intracranial aneurysms in terms of flow conditions, geometric features, and wall characteristics9,10,11,12. This study aims to describe a technique to create a new rabbit aneurysm model with both stump and bifurcation elastase-digested aneurysms in the same animal. The surgical techniques are inspired by those of Hoh et al.13 and Wanderer et al.14 with slight modifications to provide a good standardization and reproducibility and to ensure low mortality and morbidity.

<table><tbody><tr><td>4-0 resorbable suture</td><td>Ethicon Inc., USA</td><td>VCP292ZH</td><td></td></tr><tr><td>4-0 resorbable suture</td><td>Ethicon Inc., USA</td><td>VCP304H</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>Adrenaline</td><td>Amino AG</td><td>1445419</td><td>any generic</td></tr><tr><td>Amiodarone</td><td>Helvepharm AG</td><td>5078567</td><td>any generic</td></tr><tr><td>Anesthesia machine</td><td>Dr&amp;auml;ger</td><td></td><td>any other</td></tr><tr><td>Aspirin</td><td>Sanofi-Aventis (Suisse) SA</td><td>622693</td><td>any generic</td></tr><tr><td>Atropine</td><td>Labatec Pharma SA</td><td>6577083</td><td>any generic</td></tr><tr><td>Bandpass filter blue</td><td>Thorlabs</td><td>FD1B</td><td>any other</td></tr><tr><td>Bandpass filter green</td><td>Thorlabs</td><td>FGV9</td><td>any other</td></tr><tr><td>Biemer vessel clip (2x)</td><td>B. Braun Medical AG, Aesculap, Switzerland</td><td>FD560R</td><td>any other</td></tr><tr><td>Bipolar forceps</td><td></td><td></td><td>any other</td></tr><tr><td>Bispectral index (neonatal)</td><td></td><td></td><td>any other</td></tr><tr><td>Blood pressure cuff (neonatal)</td><td></td><td>any other</td><td></td></tr><tr><td>Bycilces spotlight</td><td></td><td></td><td>any other</td></tr><tr><td>Clamoxyl</td><td>GlaxoSmithKline AG</td><td>758808</td><td>any generic</td></tr><tr><td>Dexmedetomidine</td><td>Ever Pharma</td><td>136740-1</td><td>any generic</td></tr><tr><td>Elastase</td><td>Sigma Aldrich</td><td>E7885</td><td></td></tr><tr><td>Electrocardiogram electrodes</td><td></td><td></td><td></td></tr><tr><td>Ephedrine</td><td>Amino AG</td><td>1435734</td><td></td></tr><tr><td>Esmolol</td><td>OrPha Swiss GmbH</td><td>3284044</td><td></td></tr><tr><td>Fentanyl (intravenous use)</td><td>Janssen-Cilag AG</td><td>98683</td><td></td></tr><tr><td>Fentanyl (transdermal)</td><td>Mepha Pharma AG</td><td>4008286</td><td></td></tr><tr><td>Fluoresceine</td><td>Curatis AG</td><td>5030376</td><td></td></tr><tr><td>Fragmin</td><td>Pfizer PFE Switzerland GmbH</td><td>1906725</td><td></td></tr><tr><td>Heating pad or heating forced-air warming system</td><td></td><td></td><td></td></tr><tr><td>Isotonic sodium chloride solution (0.9%)</td><td>Fresenius KABI</td><td>336769</td><td></td></tr><tr><td>Ketamine</td><td>Pfizer PFE Switzerland GmbH</td><td>342261</td><td></td></tr><tr><td>lid retractor</td><td></td><td></td><td>Approach</td></tr><tr><td>Lidocaine</td><td>Streuli Pharma AG</td><td>747466</td><td></td></tr><tr><td>Longuettes</td><td></td><td></td><td></td></tr><tr><td>Metacam</td><td>Boehringer Ingelheim</td><td>P7626406</td><td>Medication</td></tr><tr><td>Methadone</td><td>Streuli Pharma AG</td><td>1084546</td><td>Sedaton</td></tr><tr><td>Micro-forceps&amp;nbsp; curved</td><td>Ulrich Swiss, Switzerland</td><td>U52-015-15</td><td></td></tr><tr><td>Micro-forceps&amp;nbsp; straight 2x</td><td>Ulrich Swiss, Switzerland</td><td>U52-010-15</td><td></td></tr><tr><td>Microscissors</td><td>Ulrich Swiss , Switzerland</td><td>U52-327-15</td><td></td></tr><tr><td>Midazolam</td><td>Accord Healthcare AG</td><td>7752484</td><td></td></tr><tr><td>Needle 23 G</td><td></td><td></td><td>arteriotomy</td></tr><tr><td>Needle holder</td><td></td><td></td><td></td></tr><tr><td>O&lt;sub&gt;2&lt;/sub&gt;-Face mask</td><td></td><td></td><td></td></tr><tr><td>Operation microscope</td><td>Wild Heerbrugg</td><td></td><td></td></tr><tr><td>Papaverin</td><td>Bichsel</td><td></td><td>topical application</td></tr><tr><td>Povidone iodine</td><td>Mundipharma Medical Company</td><td></td><td>any generic</td></tr><tr><td>Prilocaine-lidocaine creme</td><td>Emla</td><td></td><td></td></tr><tr><td>Propofol</td><td>B. Braun Medical AG, Switzerland</td><td></td><td>General anesthesia</td></tr><tr><td>Pulse oxymeter</td><td></td><td></td><td></td></tr><tr><td>Rectal temperature probe (neonatal)</td><td></td><td></td><td></td></tr><tr><td>Ringer Lactate</td><td>Bioren Sintetica SA</td><td></td><td>Infusion</td></tr><tr><td>Ropivacain</td><td>Aspen Pharma Schweiz GmbH</td><td>1882249</td><td>Local anesthesia</td></tr><tr><td>Scalpell</td><td>Swann-Morton</td><td>210</td><td></td></tr><tr><td>Small animal shaver</td><td></td><td></td><td></td></tr><tr><td>Soft tissue forceps</td><td></td><td></td><td></td></tr><tr><td>Soft tissue spreader</td><td></td><td></td><td></td></tr><tr><td>Stainless steel sponge bowls</td><td></td><td></td><td></td></tr><tr><td>Sterile micro swabs</td><td></td><td></td><td></td></tr><tr><td>Stethoscope</td><td></td><td></td><td></td></tr><tr><td>Surgery drape</td><td></td><td></td><td></td></tr><tr><td>Surgical scissors</td><td></td><td></td><td></td></tr><tr><td>Syringes 1 mL, 2 mL, and 5 mL</td><td></td><td></td><td></td></tr><tr><td>Tris-Buffer</td><td>Sigma Aldrich</td><td>93302</td><td>Elastase solution</td></tr><tr><td>Vascular clip applicator</td><td>B. Braun, Germany</td><td>FT495T</td><td></td></tr><tr><td>Vein and arterial catheter 22 G</td><td></td><td></td><td></td></tr><tr><td>vessel loop</td><td></td><td></td><td>Approach</td></tr><tr><td>video camera or smartphone</td><td></td><td></td><td></td></tr><tr><td>Vitarubin</td><td>Streuli Pharma AG</td><td>6847559</td><td></td></tr><tr><td>Yasargil titan standard clip (2x)</td><td>B. Braun Medical AG, Aesculap, Switzerland</td><td>FT242T</td><td></td></tr></tbody></table>

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.

The creation of a stump and a bifurcation aneurysm was successful in all 5 New Zealand white rabbits without intraoperative complications. No mortality was observed during surgery or during the follow-up period of 24 &#177; 2 days. One rabbit experienced postoperative complications with a vestibular syndrome and a blindness of the right side. The animal recovered completely and spontaneously after 24 h. This complication did not interfere with its normal activities (free movements, water and food intake, interactions wit...

Watch this Scientific Journal Video about Creation of Two Saccular Elastase-Digested Aneurysms with Different Hemodynamics in One Rabbit at JoVE.com

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

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 ...

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Research

JoVE Journal

Medicine

2.0K Views.  Kantonsspital Aarau. Ons protocol beschrijft een reproduceerbare techniek om een konijnenaneurysmamodel te maken met zowel stomp- als bifurcatiehemodynamiek en gedegenereerde wandconditie bij één dier en tijdens een enkele chirurgische aanpak met lage morbiditeits- en sterftecijfers en hoge doorgankelijkheidspercentages. Het model maakt een directe vergelijking mogelijk van het natuurlijke verloop van sacculaire aneurysma's met betrekking tot de rol van hemodynamiek, evenals het testen van endovasculaire appara...

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

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 ...

Creation of Murine Experimental Abdominal Aortic Aneurysms with Elastase 

Transient intraluminal infusion of porcine pancreatic elastase into the infrarenal segment of the abdominal aorta is the most widely used animal model of abdominal aortic aneurysm (AAA) ever since it was first described in rats by Anidjar and colleagues.1 The rationale for its development was based on the disrupted nature of elastin observed in AAAs. This rat model has been modified to produce AAAs in the infrarenal aortic region of mice.2 The model has the ability to add broad insight into the pathobiology of AAA due to the emergence of numerous transgenic and gene knockout mice. Moreover, it is a viable platform to test potential therapeutic agents for AAA. In this video, we demonstrate the elastase infusion AAA procedure used in our laboratory. 
Mice are anesthetized using 2.5% isoflurane, and a laparotomy is performed under sterile conditions. The abdominal aortais isolated with the assistance of an operating stereomicroscope (Leica). After placing temporary ligatures around the proximal and distal aorta, an aortotomy is created at the bifurcation with the tip of a 30-gauge needle. A heat-tapered segment of PE-10 polyethylene tubing is introduced through the aortotomy and secured. The aortic lumen is subsequently perfused for 5-15 minutes at 100 mm Hg with saline containing type I porcine pancreatic elastase (4.5 U/mL; Sigma Chemical Co.). After removing the perfusion catheter, the aortotomy is repaired without constriction of the lumen.

Creation of Murine Experimental Abdominal Aortic Aneurysms with Elastase

This video shows how to induce abdominal aortic aneurysms (AAA) in mice via transient intraluminal infusion of porcine pancreatic elastase into the infrarenal segment of the abdominal aorta. The model has the ability to add broad insight into the pathobiology of AAA due to the emergence of numerous transgenic and gene knockout mice.

Transient intraluminal infusion of porcine pancreatic elastase into the infrarenal segment of the abdominal aorta is the most widely used animal model of ...

Biology

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 ...

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 ...

Neuroscience

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. ...

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 ...

Investigating the Molecular Mechanisms Underlying Murine Abdominal Aortic Aneurysm

A Mouse Abdominal Aortic Aneurysm Model by Periadventitial Calcium Chloride and Elastase Infiltration

Abdominal aortic aneurysm (AAA) is a life-threatening disease associated with high mortality rates. It is characterized by the permanent dilation of the abdominal aorta with at least a 50% increase in arterial diameter. Various animal models of AAA have been introduced to mimic the pathophysiological changes and study the underlying mechanisms of AAA. Among these models, the calcium chloride (CaCl2)- and elastase-induced AAA models are commonly used in mice. However, these methods have certain limitations. Traditional intraluminal porcine pancreatic elastase (PPE) perfusion is associated with high technical difficulty and a high rupture rate, while periadventitial administration of PPE yields inconsistent results. In addition, the CaCl2-induced AAA model lacks human AAA features, such as atherothrombosis and aneurysm rupture. Therefore, the combined application of CaCl2 and PPE has been proposed as an approach to enhance success rates and induce greater diameter increases in AAA animal models. This manuscript presents a comprehensive protocol for establishing a mouse AAA model through periaortic infiltration of PPE and CaCl2 in the infrarenal segment of the abdominal aorta. By following this protocol, we can achieve an AAA formation rate of approximately 90% with technical simplicity and reproducibility. Further ultrasound and histological experiments confirm that this model effectively replicates the morphological and pathological changes observed in human AAA.

Author Spotlight: Development and Characterization of a Mouse Model for Abdominal Aortic Aneurysm

This manuscript presents a protocol for establishing a mouse abdominal aortic aneurysm model using calcium chloride and elastase, combining the advantages of previous modeling methods. This model can be utilized to investigate the pathophysiological mechanisms underlying abdominal aortic aneurysms.

Abdominal aortic aneurysm (AAA) is a life-threatening disease associated with high mortality rates. It is characterized by the permanent dilation of the ...

Mouse Abdominal Aortic Aneurysm Model Induced by Perivascular Application of Elastase

Abdominal aortic aneurysm (AAA), although primarily asymptomatic, is potentially life-threatening as the rupture of AAA usually has a devastating outcome. Currently, there are several distinct experimental models of AAA, each emphasizing a different aspect in the pathogenesis of AAA. The elastase-induced AAA model is the second most used rodent AAA model. This model involves direct infusion or application of porcine pancreatic elastase (PPE) to the infrarenal segment of the aorta. Due to technical challenges, most elastase-induced AAA model nowadays is performed with the external application rather than an intraluminal infusion of PPE. The infiltration of elastase will cause degradation of elastic lamellae in the medial layers, resulting in the loss of aortic wall integrity and subsequent dilation of the abdominal aorta. However, one disadvantage of the elastase-induced AAA model is the inevitable variation of how the surgery is performed. Specifically, the surgical technique of isolating the infrarenal segment of the aorta, the material used for aorta wrapping and PPE incubation, the enzymatic activity of PPE, and the time duration of PPE application can all be important determinants that affect the eventual AAA formation rate and aneurysm diameter. Notably, the difference in these factors from different studies on AAA can lead to reproducibility issues. This article describes a detailed surgical process of the elastase-induced AAA model through direct application of PPE to the adventitia of the infrarenal abdominal aorta in the mouse. Following this procedure, a stable AAA formation rate of around 80% in male and female mice is achievable. The consistency and reproducibility of AAA studies using an elastase-induced AAA model can be significantly enhanced by establishing a standard surgical procedure.

The present protocol describes a standardized surgical method for the elastase-induced AAA model through the direct application of elastase to the adventitia of infrarenal abdominal aorta in mice.

Abdominal aortic aneurysm (AAA), although primarily asymptomatic, is potentially life-threatening as the rupture of AAA usually has a devastating outcome. ...

Developing an Experimental Aneurysm Model in a Rabbit Using an Elastase-Treated Vessel Pouch

<a href='https://app.jove.com/v/61157/arterial-pouch-microsurgical-bifurcation-aneurysm-model-in-the-rabbit'>Source: Wanderer, S., et al. Arterial Pouch Microsurgical Bifurcation Aneurysm Model in the Rabbit. J. Vis. Exp. (2020)</a>This video demonstrates the creation of an experimental aneurysm model in a rabbit using an elastase-treated vessel pouch. After excising a segment of the right CCA, the pouch is treated with elastase, then sutured to the left CCA to restore blood flow. The resulting stress on the weakened pouch walls induces aneurysm formation.

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

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