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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(92), e51071 (2014).</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=Complex+bilobular,+bisaccular,+and+broad-neck+microsurgical+aneurysm+formation+in+the+rabbit+bifurcation+model+for+the+study+of+upcoming+endovascular+techniques.">Complex bilobular, bisaccular, and broad-neck microsurgical aneurysm formation in the rabbit bifurcation model for the study of upcoming endovascular techniques.</a> American Journal of Neuroradiology. 32 (4), 772-777 (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 (1), 248-254 (2014).</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=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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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&#228;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>
		</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&#160; curved</td>
			<td>Ulrich Swiss, Switzerland</td>
			<td>U52-015-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro-forceps&#160; 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>O2-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 ...

creation-two-saccular-elastase-digested-aneurysms-with-different

Research

JoVE Journal

Medicine

2.0K Views.  Kantonsspital Aarau. 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. 

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

Creation of Reversible Cholestatic Rat Model

Cholestasis is a clinical condition commonly encountered by both surgeons and gastroenterologists. Cholestasis can cause various physiological changes and affect the nutritional status and surgical outcomes. Study of the pathophysiological changes occurring in the liver and other organs is of importance. Various studies have been done in cholestatic rat models.
We used a reversible cholestatic rat model in our recent study looking at the role of methylprednisolone in the ischemia reperfusion injury. Various techniques for creation of a reversible cholestatic model have been described. Creation of a reversible cholestatic rat model can be challenging in view of the smaller size and unique hepatopancreatobiliary anatomy in rats. This video article demonstrates the creation of a reversible cholestatic model.
This model can be used in various studies, such as looking at the changes in nutritional, physiological, pathological, histological and immunological changes in the gastrointestinal tract. This model can also be used to see the effects of cholestasis and various therapeutic interventions on major hepatic surgeries.

Cholestasis is a clinical condition commonly encountered by both surgeons and gastroenterologists. Creation of a reversible cholestatic rat model can be challenging in view of the smaller size and unique hepatopancreatobiliary anatomy in rats. This video article demonstrates the creation of a reversible cholestatic model.

Cholestasis is a clinical condition commonly encountered by both surgeons and gastroenterologists. Cholestasis can cause various physiological changes and ...

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 ...

Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human pulp-derived stem cells (hDPSCs) possess high proliferation potential with multi-lineage differentiation capacity compare to the ordinary source of adult stem cells1-8; therefore, hDPSCs could be the good candidates for autologous transplantation in tissue engineering and regenerative medicine. Along with these benefits, possessing the mesenchymal stem cells (MSC) features, such as immunolodulatory effect, make hDPSCs more valuable, even in the case of allograft transplantation6,9,10. Therefore, the primary step for using this source of stem cells is to select the best protocol for isolating hDPSCs from pulp tissue. In order to achieve this goal, it is crucial to investigate the effect of various isolation conditions on different cellular behaviors, such as their common surface markers &amp; also their differentiation capacity.
Thus, here we separate human pulp tissue from impacted third molar teeth, and then used both existing protocols based on literature, for isolating hDPSCs,11-13 i.e. enzymatic dissociation of pulp tissue (DPSC-ED) or outgrowth from tissue explants (DPSC-OG). In this regards, we tried to facilitate the isolation methods by using dental diamond disk. Then, these cells characterized in terms of stromal-associated Markers (CD73, CD90, CD105 &amp; CD44), hematopoietic/endothelial Markers (CD34, CD45 &amp; CD11b), perivascular marker, like CD146 and also STRO-1. Afterwards, these two protocols were compared based on the differentiation potency into odontoblasts by both quantitative polymerase chain reaction (QPCR) &amp; Alizarin Red Staining. QPCR were used for the assessment of the expression of the mineralization-related genes (alkaline phosphatase; ALP, matrix extracellular phosphoglycoprotein; MEPE &amp; dentin sialophosphoprotein; DSPP).14

The method described isolation and characterization of human Dental Pulp Stem Cells (hDPSCs) by using either enzymatic dissociation of pulp (DPSC-ED) or direct outgrowth of stem cells from pulp tissue explants (DPSC-OG). Then followed by in vitro comparative differentiation of both types of hDPSCs into odontoblasts.

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human ...

Non-invasive Optical Measurement of Cerebral Metabolism and Hemodynamics in Infants

Perinatal brain injury remains a significant cause of infant mortality and morbidity, but there is not yet an effective bedside tool that can accurately screen for brain injury, monitor injury evolution, or assess response to therapy. The energy used by neurons is derived largely from tissue oxidative metabolism, and neural hyperactivity and cell death are reflected by corresponding changes in cerebral oxygen metabolism (CMRO2). Thus, measures of CMRO2 are reflective of neuronal viability and provide critical diagnostic information, making CMRO2 an ideal target for bedside measurement of brain health.
Brain-imaging techniques such as positron emission tomography (PET) and single-photon emission computed tomography (SPECT) yield measures of cerebral glucose and oxygen metabolism, but these techniques require the administration of radionucleotides, so they are used in only the most acute cases.
Continuous-wave near-infrared spectroscopy (CWNIRS) provides non-invasive and non-ionizing radiation measures of hemoglobin oxygen saturation (SO2) as a surrogate for cerebral oxygen consumption. However, SO2 is less than ideal as a surrogate for cerebral oxygen metabolism as it is influenced by both oxygen delivery and consumption. Furthermore, measurements of SO2 are not sensitive enough to detect brain injury hours after the insult 1,2, because oxygen consumption and delivery reach equilibrium after acute transients 3. We investigated the possibility of using more sophisticated NIRS optical methods to quantify cerebral oxygen metabolism at the bedside in healthy and brain-injured newborns. More specifically, we combined the frequency-domain NIRS (FDNIRS) measure of SO2 with the diffuse correlation spectroscopy (DCS) measure of blood flow index (CBFi) to yield an index of CMRO2 (CMRO2i) 4,5.
With the combined FDNIRS/DCS system we are able to quantify cerebral metabolism and hemodynamics. This represents an improvement over CWNIRS for detecting brain health, brain development, and response to therapy in neonates. Moreover, this method adheres to all neonatal intensive care unit (NICU) policies on infection control and institutional policies on laser safety. Future work will seek to integrate the two instruments to reduce acquisition time at the bedside and to implement real-time feedback on data quality to reduce the rate of data rejection.

We combined frequency-domain near-infrared spectroscopy measures of cerebral hemoglobin oxygenation with diffuse correlation spectroscopy measures of cerebral blood flow index to estimate an index of oxygen metabolism. We tested the utility of this measure as a bedside screening tool to evaluate the health and development of the newborn brain.

Perinatal brain injury remains a significant cause of infant mortality  and morbidity, but there is not yet an effective bedside tool that can  accurately ...

Subcutaneous Angiotensin II Infusion using Osmotic Pumps Induces Aortic Aneurysms in Mice

Osmotic pumps continuously deliver compounds at a constant rate into small animals. This article introduces a standard protocol used to induce aortic aneurysms via subcutaneous infusion of angiotensin II (AngII) from implanted osmotic pumps. This protocol includes calculation of AngII amount and dissolution, osmotic pump filling, implantation of osmotic pumps subcutaneously, observation after pump implantation, and harvest of aortas to visualize aortic aneurysms in mice. Subcutaneous infusion of AngII through osmotic pumps following this protocol is a reliable and reproducible technique to induce both abdominal and thoracic aortic aneurysms in mice. Infusion durations range from a few days to several months based on the purpose of the study. AngII 1,000 ng/kg/min is sufficient to provide maximal effects on abdominal aortic aneurysmal formation in male hypercholesterolemic mouse models such as apolipoprotein E deficient or low-density lipoprotein receptor deficient mice. Incidence of abdominal aortic aneurysms induced by AngII infusion via osmotic pumps is 5 - 10 times lower in female hypercholesterolemic mice and also lower in both genders of normocholesterolemic mice. In contrast, AngII-induced thoracic aortic aneurysms in mice are not hypercholesterolemia or gender-dependent. Importantly, multiple features of this mouse model recapitulate those of human aortic aneurysms.

Subcutaneous implantation of osmotic pumps provides a convenient approach for prolonged and consistent delivery of compounds. This approach has been used extensively to study both abdominal and thoracic aortic aneurysms in mice.

Osmotic pumps continuously deliver compounds at a constant rate into small animals.  This article introduces a standard protocol used to induce aortic ...

Creation of Abdominal Adhesions in Mice

Abdominal adhesions consist of fibrotic tissue that forms in the peritoneal space in response to an inflammatory insult, typically surgery or intraabdominal infection. The precise mechanisms underlying adhesion formation are poorly understood. Many compounds and physical barriers have been tested for their ability to prevent adhesions after surgery with varying levels of success. The mouse and rat are important models for the study of abdominal adhesions. Several different techniques for the creation of adhesions in the mouse and rat exist in the literature. Here we describe a protocol utilizing abrasion of the cecum with sandpaper and sutures placed in the right abdominal sidewall. The mouse is anesthetized and the abdomen is prepped. A midline laparotomy is created and the cecum is identified. Sandpaper is used to gently abrade the surface of the cecum. Next, several figure-of-eight sutures are placed into the peritoneum of the right abdominal sidewall. The abdominal cavity is irrigated, a small amount of starch is applied, and the incision is closed. We have found that this technique produces the most consistent adhesions with the lowest mortality rate.

Abdominal adhesions that form after surgery are a major cause of pain, infertility, and hospitalization and reoperation for small bowel obstruction. Our surgical procedure for creating abdominal adhesions in mice is a reliable tool to study the mechanisms underlying the formation of adhesions.

Abdominal adhesions consist of fibrotic tissue that forms in the peritoneal space in response to an inflammatory insult, typically surgery or ...

Treatment with Vancomycin Loaded Calcium Sulphate and Autogenous Bone in an Improved Rabbit Model of Bone Infection

Bone infection results from bacterial invasion, which is extremely difficult to treat in clinical, orthopedic, and traumatic surgery. The bone infection may result in sustained inflammation, osteomyelitis, and eventual bone non-union. Establishment of a feasible, reproducible animal model is important to bone infection research and antibiotic treatment. As an in vivo model, the rabbit model is widely used in bone infection research. However, previous studies on rabbit bone infection models showed that the infection status was inconsistent, as the amount of bacteria was variable. This study presents an improved surgical method for inducing bone infection on a rabbit, by blocking the bacteria in the bone marrow. Then, multi-level evaluations can be carried out to verify the modelling method.
In general, debriding necrotic tissue and implantation of vancomycin-loaded calcium sulphate (VCS) are predominant in antibiotic treatment. Although calcium sulphate in VCS benefits osteocyte crawling and new bone growth, massive bone defects occur after debriding. Autogenous bone (AB) is an appealing strategy to overcome bone defects for the treatment of massive bone defects after debriding necrotic bone.
In this study, we used the tail bone as an autogenous bone implanted in the bone defect. Bone repair was measured using micro-computed-tomography (micro-CT) and histological analysis after animal sacrifice. As a result, in the VCS group, bone non-union was consistently obtained. In contrast, the bone defect areas in the VCS-AB group were decreased significantly. The present modeling method described a reproducible, feasible, stable method to prepare a bone infection model. The VCS-AB treatment resulted in lower bone non-union rates after antibiotic treatment. The improved bone infection model and the combination treatment of VCS and autogenous bone could be helpful in studying the underlying mechanisms in bone infection and bone regeneration pertinent to traumatology orthopedic applications.

This study presents an improved rabbit model infected with Staphylococcus aureus by blocking the same amount of bacteria in bone marrow. Vancomycin loaded calcium sulphate and autogenous bone are used for antibiotic and bone repair treatment. The protocol could be helpful for studying bone infection and regeneration.

Bone infection results from bacterial invasion, which is extremely difficult to treat in clinical, orthopedic, and traumatic surgery. The bone infection ...

Creation of Colonic Anastomosis in Mice

Intestinal anastomoses are commonly performed in both elective and emergent operations. Even so, anastomotic leaks are a highly feared complications of colonic surgeries and can occur in up to 26% of surgical anastomoses, with mortality being up to 39% for patients with such a leak. Currently, there remains a paucity of data detailing the cellular mechanisms of anastomotic healing. Devising preventative strategies and treatment modalities for anastomotic leak could be greatly potentiated by a better understanding of appropriate anastomotic healing.
A murine model is ideal as previous studies have shown that the murine anastomosis is the most clinically similar to the human case as compared with other animal models. We offer an easily reproducible murine model of colonic anastomosis in mice that will allow for further illustration of anastomotic healing.

Anastomotic leak or breakdown after surgery is a major cause of postoperative morbidity and mortality. Our surgery for creating a colonic anastomosis is a reliable and reproducible method for studying the healing mechanisms of anastomoses.

Intestinal anastomoses are commonly performed in both elective and emergent operations. Even so, anastomotic leaks are a highly feared complications of ...

A Modified Two Kidney One Clip Mouse Model of Renin Regulation in Renal Artery Stenosis

Renal artery stenosis is a common condition in patients with coronary or peripheral vascular disease where the renin angiotensin aldosterone system (RAAS) is overactivated. In this context, there is a narrowing of the renal arteries that stimulate an increase in the expression and release of renin, the rate-limiting protease in RAAS. The resulting rise in renin expression is a known driver of renovascular hypertension, frequently associated with kidney injury and end organ damage. Thus, there is a great interest in developing novel treatments for this condition. The molecular and cellular mechanism of renin control in renal artery stenosis is not fully understood and warrants further investigation. To induce renal artery stenosis in mice, a modified 2 kidney 1 clip (2K1C) Goldblatt mouse model was developed. The right kidney was stenosed in wild type mice and sham operated mice were used as control. After renal artery stenosis, we determined renin expression and kidney injury. Kidneys were harvested, and fresh cortices were used to determine protein and mRNA expression of renin. This animal model is reproducible and can be used to study pathophysiological responses, molecular and cellular pathways involved in renovascular hypertension and kidney injury.

A modified 2 kidney 1 clip (2K1C) Goldblatt mouse model was developed using polyurethane tubing to initiate renal artery stenosis, inducing an increase in renin expression and kidney injury. Here, we describe a detailed procedure of preparing and placing the cuff onto the renal artery to generate a reproducible and consistent 2K1C mouse model.

Renal artery stenosis is a common condition in patients with coronary or peripheral vascular disease where the renin angiotensin aldosterone system (RAAS) ...

Electrocardiogram Recordings in Anesthetized Mice using Lead II

The electrocardiogram is a valuable tool for evaluating the cardiac conduction system. Animal research has helped generate novel genetic and pharmacological information regarding the electrocardiogram. However, making electrocardiogram measurements in small animals in vivo, such as mice, has been challenging. To this end, we used an electrocardiogram recording method in anesthetized mice with many advantages: it is a technically simple procedure, is inexpensive, has short measuring time, and is affordable, even in young mice. Despite the limitations with using anesthesia, comparisons between control and experimental groups can be performed with enhanced sensitivity. We treated mice with agonists and antagonists of the autonomic nervous system to determine the validity of this protocol and compared our results with previous reports. Our ECG protocol detected increased heart rates and QTc intervals on treatment with atropine, decreased heart rates and QTc intervals after carbachol treatment, and higher heart rates and QTc intervals with isoprenaline but did not note any change in ECG parameters on administration of propranolol. These results are supported by previous reports, confirming the reliability of this ECG protocol. Thus, this method can be used as a screening approach to making ECG measurements that otherwise would not be attempted due to high cost and technical difficulties.

We present an ECG protocol that is technically easy, inexpensive, fast, and affordable in small mice, and can be performed with enhanced sensitivity. We suggest this method as a screening approach for studying pharmacological agents, genetic modifications, and disease models in mice.