We are grateful to Professor Heber Ferraz Leite, the Director of so many international microsurgical workshops around the world, for his open-minded and valuable teaching culture.
We acknowledge support from the Open Access Publishing Fund of Karl Landsteiner University of Health Sciences, Krems, Austria. This study was funded by a grant from the Scientific Fund of the Mayor of Vienna. The cost of this publication was funded by the Open Access Publishing Fund of Karl Landsteiner University of Health Sciences, Krems, Austria. The funding bodies played no role in the design of the study, the collection, analysis, and interpretation of the data, and the writing of the manuscript.

The animal studies were approved by the Institute Animal Ethics Committee of the institute at which this study was conducted. For this animal model, New Zealand white rabbits (2.5-5.5 kg) were used.
NOTE: Our standard technique for the creation of the venous pouch true arterial bifurcation aneurysms in rabbits was published in 2011, and an adaption for giant aneurysms was published in 201612,13. We summarize these techniques and highlight essential steps for the modification of giant aneurysms.
1. Preoperative phase
<ol>
	<li>Administer ketamine (30 mg/kg) and xylazine (6 mg/kg) via perilumbar intramuscular injection for general anesthesia. Then, intubate the rabbit (tube diameter: 4 mm, length: 18 mm; this size may vary depending&#160;on the animal&#39;s size), and continue with gas anesthesia (2% isoflurane). Monitor the depth of anesthesia by a toe pinch every 15 min, and adjust if necessary.</li>
	<li>Shave the area from the angle of the jaw down to the thorax using clippers. Disinfect the surgical area using at least three alternating rounds of chlorhexidine or povidone-iodine scrub followed by alcohol. Drape the surgical site.</li>
</ol>
2. Surgical phase I
<ol>
	<li>Incise the skin along the midline from the angle of the jaw down to the manubrium sterni using a scalpel. Perform blunt dissection in the subcutis.</li>
	<li>Switch to the surgical microscope. Dissect a 2-3 cm long branchless segment of the left external jugular vein. Apply 4% papaverine dropwise repeatedly on the vessels to prevent vasospasm and optionally add 5 mg/mL neomycin sulfate dropwise for infection control.</li>
	<li>Harvest the vein segment after proximal and distal ligation using 6-0 non-resorbable sutures. Put the vein segment in a heparinized saline solution (1,000 IU heparin in 20 mL of 0.9% saline and 1 mL of 4% papaverine HCl)13.</li>
</ol>
3. Surgical phase II
<ol>
	<li>Prepare both CCAs by dissecting them from the carotid bifurcation down to their origin. Watch carefully for the medial arterial branches, which supply the laryngeal, tracheal, and neural structures.</li>
	<li>Administer 1,000 IU heparin intravenously.</li>
	<li>Apply a temporal microsurgical clip at the distal end of the right CCA.</li>
	<li>Ligate and cut the right CCA proximally directly above the brachiocephalic trunk using poly filament 6-0 non-resorbable sutures.</li>
	<li>Use a sterile piece of rubber (e.g. from a glove)&#160;as an underlay to facilitate the procedure. Remove the adventitia at the anastomosis site of both vessels with anatomic micro-forceps and micro-scissors. Clip the anastomosis site of the left CCA distally and proximally13.</li>
</ol>
4. Surgical phase III
<ol>
	<li>Make an arteriotomy at the left CCA according to the size of the planned anastomosis with the right CCA and the venous pouch. Determine the length of the arteriotomy by the diameter of the contralateral carotid artery (about 2 mm) together with the size of the planned aneurysm neck. 
	NOTE: The size is as flexible as the possible aneurysm sizes and neck sizes of this universal aneurysm model. The minimum size should not be smaller than 3 mm and can be up to a maximum of about 15 mm.</li>
	<li>Clean the aneurysm site with heparinized saline (about 5 mL). Using four to five non-resorbable 10-0 monofilament sutures, and suture the posterior circumference of the right CCA stump with the before-described arteriotomy of the left CCA.</li>
	<li>Cut the stump of the right CCA longitudinally to a length of 1-1.5 cm. Anastomose the posterior part of the venous pouch with the arteriotomy of the left CCA using 10-0 sutures. Then, suture the rear side of the venous pouch with the posterior wall of the right CCA with three to four sutures.</li>
	<li>Suture the anterior anastomosis in the same sequence.</li>
	<li>Release the temporal clip on the right CCA. Usually, the anastomosis leaks. Use this to wash air and blood clots out.</li>
	<li>Seal the anastomosis with fat derived from the subcutaneous tissue of the surgical approach and fibrin glue.</li>
	<li>Close the fascia using 4-0 non-resorbable sutures. Perform the wound closure using 4-0 resorbable sutures13.</li>
</ol>
5. Postoperative phase
<ol>
	<li>Administer 10 mg/kg acetylsalicylic acid intravenously.</li>
	<li>Achieve postoperative analgesia by a transdermal fentanyl patch (12.5. &#181;g/h) at the shaved region for 3 days13.&#160; 
	NOTE:&#160;Consult the facility veterinarian on appropriate 
	analgesia options.</li>
	<li>Achieve postoperative anticoagulation by administering 100 IU/kg low molecular heparin daily subcutaneously for 2 weeks.</li>
</ol>

<ol>
	<li>Molyneux, A. 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=International+subarachnoid+aneurysm+trial+(ISAT)+of+neurosurgical+clipping+versus+endovascular+coiling+in+2143+patients+with+ruptured+intracranial+aneurysms:+A+randomised+comparison+of+effects+on+survival,+dependency,+seizures,+rebleeding,+subgroups,+and+aneurysm+occlusion.">International subarachnoid aneurysm trial (ISAT) of neurosurgical clipping versus endovascular coiling in 2143 patients with ruptured intracranial aneurysms: A randomised comparison of effects on survival, dependency, seizures, rebleeding, subgroups, and aneurysm occlusion.</a> Lancet. 366 (9488), 809-817 (2005).</li><li>Algra, A. M., 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=Procedural+clinical+complications,+case-fatality+risks,+and+risk+factors+inendovascular+and+neurosurgical+treatment+of+unruptured+intracranial+aneurysms:+A+systematic+review+and+meta-analysis.">Procedural clinical complications, case-fatality risks, and risk factors inendovascular and neurosurgical treatment of unruptured intracranial aneurysms: A systematic review and meta-analysis.</a> JAMA Neurology. 76 (3), 282-293 (2019).</li><li>Laurent, D., 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=The+evolution+of+endovascular+therapy+for+intracranial+aneurysms:+Historical+perspective+and+next+frontiers.">The evolution of endovascular therapy for intracranial aneurysms: Historical perspective and next frontiers.</a> Neuroscience Insights. 17, (2022).</li><li>B&#246;cher-Schwarz, H. G., 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=Histological+findings+in+coil-packed+experimental+aneurysms+3+months+after+embolization.">Histological findings in coil-packed experimental aneurysms 3 months after embolization.</a> Neurosurgery. 50 (2), 375-379 (2002).</li><li>Sherif, C., Plenk, H. J., Grossschmidt, K., Kanz, F., Bavinzski, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Computer-assisted+quantification+of+occlusion+and+coil+densities+on+angiographic+and+histological+images+of+experimental+aneurysms.">Computer-assisted quantification of occlusion and coil densities on angiographic and histological images of experimental aneurysms.</a> Neurosurgery. 58 (3), 559-566 (2006).</li><li>Massoud, T. F., Guglielmi, G., Ji, C., Vi&#241;uela, F., Duckwiler, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+saccular+aneurysms.+I.+Review+of+surgically-constructed+models+and+their+laboratory+applications.">Experimental saccular aneurysms. I. Review of surgically-constructed models and their laboratory applications.</a> Neuroradiology. 36 (7), 537-546 (1994).</li><li>Anidjar, 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=Elastase-induced+experimental+aneurysms+in+rats.">Elastase-induced experimental aneurysms in rats.</a> Circulation. 82 (3), 973-981 (1990).</li><li>Wakhloo, A. K., Schellhammer, F., de Vries, J., Haberstroh, J., Schumacher, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-expanding+and+balloon-expandable+stents+in+the+treatment+of+carotid+aneurysms:+An+experimental+study+in+a+canine+model.">Self-expanding and balloon-expandable stents in the treatment of carotid aneurysms: An experimental study in a canine model.</a> AJNR. American Journal of Neuroradiology. 15 (3), 493-502 (1994).</li><li>Dai, D., 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=Histopathologic+and+immunohistochemical+comparison+of+human,+rabbit,+and+swine+aneurysms+embolized+with+platinum+coils.">Histopathologic and immunohistochemical comparison of human, rabbit, and swine aneurysms embolized with platinum coils.</a> American Journal of Neuroradiology. 26 (10), 2560-2568 (2005).</li><li>Shin, Y. 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=Creation+of+four+experimental+aneurysms+with+different+hemodynamics+in+one+dog.">Creation of four experimental aneurysms with different hemodynamics in one dog.</a> American Journal of Neuroradiology. 26 (7), 1764-1767 (2005).</li><li>Abruzzo, T., 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=Histologic+and+morphologic+comparison+of+experimental+aneurysms+with+human+intracranial+aneurysms.">Histologic and morphologic comparison of experimental aneurysms with human intracranial aneurysms.</a> AJNR. American Journal of Neuroradiology. 19 (7), 1309-1314 (1998).</li><li>Sherif, C., Plenk, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+angiographic+and+histopathologic+evaluation+of+experimental+aneurysms.">Quantitative angiographic and histopathologic evaluation of experimental aneurysms.</a> American Journal of Neuroradiology. 32 (2), 33 (2011).</li><li>Sherif, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microsurgical+venous+pouch+arterial-bifurcation+aneurysms+in+the+rabbit+model:+Technical+aspects.">Microsurgical venous pouch arterial-bifurcation aneurysms in the rabbit model: Technical aspects.</a> Journal of Visualized Experiments. (51), e2718 (2011).</li><li>Sherif, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Very+large+and+giant+microsurgical+bifurcation+aneurysms+in+rabbits:+Proof+of+feasibility+and+comparability+using+computational+fluid+dynamics+and+biomechanical+testing.">Very large and giant microsurgical bifurcation aneurysms in rabbits: Proof of feasibility and comparability using computational fluid dynamics and biomechanical testing.</a> Journal of Neuroscience Methods. 268, 7-13 (2016).</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=Long-term+patency+of+complex+bilobular,+bisaccular,+and+broad-neck+aneurysms+in+the+rabbit+microsurgical+venous+pouch+bifurcation+model.">Long-term patency of complex bilobular, bisaccular, and broad-neck aneurysms in the rabbit microsurgical venous pouch bifurcation model.</a> Neurological Research. 34 (6), 538-546 (2012).</li><li>Sherif, C., Marbacher, S., Erhardt, S., Fandino, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+microsurgical+creation+of+venous+pouch+arterial+bifurcation+aneurysms+in+rabbits.">Improved microsurgical creation of venous pouch arterial bifurcation aneurysms in rabbits.</a> American Journal of Neuroradiology. 32 (1), 165-169 (2011).</li><li>Spetzger, U., 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=Microsurgically+produced+bifurcation+aneurysms+in+a+rabbit+model+for+endovascular+coil+embolization.">Microsurgically produced bifurcation aneurysms in a rabbit model for endovascular coil embolization.</a> Journal of Neurosurgery. 85 (3), 488-495 (1996).</li><li>Bavinzski, G., 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=Experimental+bifurcation+aneurysm:+A+model+for+in+vivo+evaluation+of+endovascular+techniques.">Experimental bifurcation aneurysm: A model for in vivo evaluation of endovascular techniques.</a> Minimally invasive neurosurgery. 41 (3), 129-132 (1998).</li><li>Forrest, M. D., O'Reilly, G. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+experimental+aneurysms+at+a+surgically+created+arterial+bifurcation.">Production of experimental aneurysms at a surgically created arterial bifurcation.</a> American Journal of Neuroradiology. 10 (2), 400-402 (1989).</li><li>Ysuda, R., Strother, C. M., Aagaard-Kienitz, B., Pulfer, K., Consigny, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+large+and+giant+bifurcation+aneurysm+model+in+canines:+proof+of+feasibility.">A large and giant bifurcation aneurysm model in canines: proof of feasibility.</a> American Journal of Neuroradiology. 33 (3), 507-512 (2012).</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>Sherif, C., Marbacher, S., Fandino, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Computerized+angiographic+evaluation+of+coil+density+and+occlusion+rate+in+embolized+cerebral+aneurysms.">Computerized angiographic evaluation of coil density and occlusion rate in embolized cerebral aneurysms.</a> Acta Neurochirurgica. 153 (2), 343-344 (2011).</li></ol>

The authors have no relevant financial or non-financial interests to disclose.

There are some critical steps to ensure the replicability of the protocol described above. The meticulous removal of the thrombogenic periadventitial tissue at the anastomosis site is essential13. One must ensure that the anastomosis is tensionless and has as few sutures as possible. For giant aneurysms, it is important to begin with the back side of the anastomosis. This gives better sight and control for the most challenging sutures as compared to previously proposed procedures17,18,19.
Contrary to normal-sized aneurysms, the key factor for the retrieval of the venous pouch is the meticulous preparation of a 2-3 cm long vein segment. It is crucial to dissect all the small side branches of the external jugular vein to be able to safely ligate them. While suturing the anastomoses, direct contact with the vessels should be avoided by leaving the ends of the single sutures a bit longer. Only these free suture ends should be grabbed with the forceps to move the aneurysm complex. This technical detail helps in the use of a no-touch technique with the vessels, which is a general principle in vascular microsurgery. Another challenge, as compared to normal-sized aneurysms, is the impaired sight to the back side of the vessel aneurysm complex caused by the giant aneurysm sac. This can lead to increased technical difficulties at the rear side of the anastomosis. After completing the anastomosis, a longer flushing time is necessary due to the higher thrombus formation probability within the giant aneurysm sac. One should be aware of leakages, as they are very common. If they are not sealed with the fat pad, additional sutures should be performed.
A limitation is the use of an extracranial aneurysm as a model for intracranial pathology. Furthermore, high microsurgical requirements and well-equipped laboratories are needed for the successful implementation of this protocol. Also, rabbits are sensitive animals, and good animal housing is crucial for survival rates.
The presented model offers several advantages over the current widely used models. The most widespread current model for cerebral aneurysms is the elastase model. However, for this model, biomechanical testing of the aneurysm wall properties has never been performed. Therefore, the biomechanical comparability of this model to human conditions is unclear. On the contrary, this biomechanical testing is available for our proposed model, showing good comparability to human conditions14. Another significant advantage of this proposed model over the elastase model is the true bifurcational hemodynamics18. This model is created in a true artificially created bifurcation, while the elastase-digested aneurysm sac is formed at the dead end of the CCA, more or less mimicking a sidewall geometry.
Up to this date, there were nearly no other giant aneurysm models available. However, these models are strongly needed for the evaluation of new endovascular devices. Going through the literature, only one canine model for giant bifurcation aneurysms has been described20. However, canine hemodynamics and the coagulation system showed significant differences in comparison to humans, whereas the rabbit model has shown its superiority regarding its comparability to humans14.
Newly developed endovascular devices for aneurysm treatment are commonly tested in rabbit models. Our previously published venous pouch bifurcation aneurysms&#160;model has been used for the CE and FDA approval of such devices3,18. However, a reliable and comparable animal model for giant aneurysms in rabbits was not available until recently. In humans, giant aneurysms have the highest rates of recanalization and delayed rupture after endovascular treatment. Therefore, new endovascular devices are urgently wanted, and the industry has brought up the need for a giant aneurysm rabbit model. Another application is the evaluation of the aneurysm wall using high-field magnetic resonance imaging, which aims to identify potential risk factors for rupture, such as aneurysm wall diameter or contrast enhancement behavior22. Furthermore, long-term studies are needed to evaluate the patency of this aneurysm model over time, as well as studies showing the aneurysm behavior with flow diverter stents and intrasaccular flow diverters.

Endovascular embolization has become an important alternative to aneurysm clipping for the treatment of ruptured cerebral aneurysms1. The main drawback of this treatment strategy is the high rates of aneurysm recanalization with delayed aneurysm rupture2. Large and giant aneurysms have been shown to be especially prone to these complications. Therefore, new endovascular devices are constantly being developed3. Models for experimental studies are essential for testing these devices4,5.
Human cerebral aneurysms have been studied in rats, rabbits, canines, and swine6,7,8. However, rabbit models have shown the best comparability to humans regarding hemodynamics and the coagulation system9,10,11,12. In the venous pouch arterial bifurcation model in rabbits, a venous pouch is sutured into a microsurgically created true bifurcation of both common carotid arteries (CCA) to mimic an aneurysm13. However, a true bifurcation model for giant aneurysms in rabbits was not available until recently. The first results using computational fluid dynamics and biomechanical testing were published by our group in 201614.
As giant aneurysms represent challenging lesions for treatment in humans and a reliable animal model is crucial for their research, we present a condensed summary of the improved techniques for the creation of giant experimental aneurysms12,13. The advantages of using this method are (i) the minimal morbidity and high aneurysm patency rates14, high comparability to humans regarding hemodynamics and the coagulation system9,10,11,12, and cost-effectiveness compared to canine methods, (ii) the true bifurcation design for a giant aneurysm13, (iii) the good hemodynamic comparability of the created aneurysms shown by computational fluid dynamics14, and (iv) the high long-term patency rates15.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.9% Saline</td>
			<td></td>
			<td></td>
			<td>Any genericon</td>
		</tr>
		<tr>
			<td>4% Papaverin HCl</td>
			<td></td>
			<td></td>
			<td>Any genericon</td>
		</tr>
		<tr>
			<td>Ethilon 10-0 monofil non resorbable sutures&#160;</td>
			<td>Ethicon Inc</td>
			<td>2814</td>
			<td>Taper point needle</td>
		</tr>
		<tr>
			<td>Evicel Bioglue&#160;</td>
			<td>Ethicon Biosurgery Inc.</td>
			<td>3901</td>
			<td></td>
		</tr>
		<tr>
			<td>Fentanyl dermal patch 12.5 &#956;g/h</td>
			<td></td>
			<td></td>
			<td>Any genericon</td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td></td>
			<td></td>
			<td>Any genericon</td>
		</tr>
		<tr>
			<td>Ketamin 50 mg/mL</td>
			<td></td>
			<td></td>
			<td>Any genericon</td>
		</tr>
		<tr>
			<td>Neomycin sulfate 5 mg/mL</td>
			<td></td>
			<td></td>
			<td>Any genericon</td>
		</tr>
		<tr>
			<td>Vicryl 4-0 polyfilament restorable sutures&#160;</td>
			<td>Ethicon Inc</td>
			<td>J386H</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylazine 20 mg/mL</td>
			<td></td>
			<td></td>
			<td>Any genericon</td>
		</tr>
	</tbody>
</table>

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.

In 2011, we published an improved technique for the venous pouch arterial bifurcation model for the creation of aneurysms in rabbits16. The mean aneurysm length was 7.9 mm, and the mean neck width was 4.1 mm. By using interrupted suture and aggressive anticoagulation, we were able to achieve 0% mortality and patency in 14 of 16 aneurysms. This technique was then adapted for the creation of giant aneurysms, and computational fluid dynamics and biomechanical testing were performed in 201614. In this study, the anesthologic management was also changed from the use of ventilation masks to intubation due to the availability of an experienced veterinarian. This represents a critical step in our experience, as the intubation of a rabbit can be difficult and lead to high preoperative mortality rates. Furthermore, the postoperative anticoagulation with low molecular heparin was reduced from 250 IU/kg to 100 IU/kg. With this regime, we were able to achieve 0% mortality and patency in 11 of 12 aneurysms. The aneurysm lengths were 21.5-25.6 mm, with neck widths from 7.3-9.8 mm. Detailed results of this study are shown in Table 1. Furthermore, these aneurysms were used for the evaluation of endovascular devices. An image of a stent-assisted embolized giant aneurysm after aneurysm retrieval is shown in Figure 1.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63738/63738fig01.jpg" /> 
Figure 1: Photo of a stent-assisted embolized giant aneurysm after aneurysm retrieval. 1 left CCA, stented parent vessels; 2 right CCA, parent vessel; + embolized aneurysm sac. <a href="https://www.jove.com/files/ftp_upload/63738/63738fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Aneurysm No.</td>
			<td>Patency</td>
			<td>Parent artery diameter [mm]</td>
			<td>Length [mm]</td>
			<td>Neck width [mm]</td>
			<td>Dome width [mm]</td>
			<td>Aspect-ratio [-]</td>
		</tr>
		<tr>
			<td>2</td>
			<td>no</td>
			<td>&#160;--</td>
			<td>--</td>
			<td>--</td>
			<td>--</td>
			<td>--</td>
		</tr>
		<tr>
			<td>1</td>
			<td>yes</td>
			<td>2.4</td>
			<td>23.4</td>
			<td>7.7</td>
			<td>9.9</td>
			<td>3</td>
		</tr>
		<tr>
			<td>3</td>
			<td>yes</td>
			<td>2.2</td>
			<td>25.1</td>
			<td>8.7</td>
			<td>10.3</td>
			<td>2.9</td>
		</tr>
		<tr>
			<td>4</td>
			<td>yes</td>
			<td>2.5</td>
			<td>23.5</td>
			<td>9.8</td>
			<td>10.6</td>
			<td>2.4</td>
		</tr>
		<tr>
			<td>5</td>
			<td>yes</td>
			<td>2.8</td>
			<td>24.8</td>
			<td>8.6</td>
			<td>9.8</td>
			<td>2.9</td>
		</tr>
		<tr>
			<td>6</td>
			<td>yes</td>
			<td>2.5</td>
			<td>21.5</td>
			<td>9.8</td>
			<td>9.3</td>
			<td>2.2</td>
		</tr>
		<tr>
			<td>7</td>
			<td>yes</td>
			<td>2.2</td>
			<td>24.2</td>
			<td>7.9</td>
			<td>10.5</td>
			<td>3.1</td>
		</tr>
		<tr>
			<td>8</td>
			<td>yes</td>
			<td>2.3</td>
			<td>25.6</td>
			<td>9.3</td>
			<td>10.2</td>
			<td>2.8</td>
		</tr>
		<tr>
			<td>9</td>
			<td>yes</td>
			<td>2.4</td>
			<td>22.1</td>
			<td>7.3</td>
			<td>10</td>
			<td>3</td>
		</tr>
		<tr>
			<td>10</td>
			<td>yes</td>
			<td>2.2</td>
			<td>25.6</td>
			<td>8.9</td>
			<td>9.7</td>
			<td>2.9</td>
		</tr>
		<tr>
			<td>11</td>
			<td>yes</td>
			<td>2.3</td>
			<td>23.4</td>
			<td>9.7</td>
			<td>11.1</td>
			<td>2.4&#160;</td>
		</tr>
	</tbody>
</table>
Table 1:&#160;Aneurysm data generated for computational fluid dynamics and biomechanical testing. The updated and detailed results of 11 aneurysms created in 2016 are shown. This table has been modified from Sherif et al.14.

Watch this Scientific Journal Video about Microsurgical Creation of Giant Bifurcation Aneurysms in Rabbits for the Evaluation of Endovascular Devices at JoVE.com

Microsurgical Creation of Giant Bifurcation Aneurysms in Rabbits for the Evaluation of 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 ...

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Research

JoVE Journal

Neuroscience

613 Views.  Karl Landsteiner University of Health Sciences. Here, we describe the technique for the microsurgical creation of giant bifurcation aneurysms in rabbits for the evaluation of endovascular devices. 

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

Focal Cerebral Ischemia Model by Endovascular Suture Occlusion of the Middle Cerebral Artery in the Rat

Stroke is the leading cause of disability and the third leading cause of death in adults worldwide1. In human stroke, there exists a highly variable clinical state; in the development of animal models of focal ischemia, however, achieving reproducibility of experimentally induced infarct volume is essential. The rat is a widely used animal model for stroke due to its relatively low animal husbandry costs and to the similarity of its cranial circulation to that of humans2,3. In humans, the middle cerebral artery (MCA) is most commonly affected in stroke syndromes and multiple methods of MCA occlusion (MCAO) have been described to mimic this clinical syndrome in animal models. Because recanalization commonly occurs following an acute stroke in the human, reperfusion after a period of occlusion has been included in many of these models. In this video, we demonstrate the transient endovascular suture MCAO model in the spontaneously hypertensive rat (SHR). A filament with a silicon tip coating is placed intraluminally at the MCA origin for 60 minutes, followed by reperfusion. Note that the optimal occlusion period may vary in other rat strains, such as Wistar or Sprague-Dawley. Several behavioral indicators of stroke in the rat are shown. Focal ischemia is confirmed using T2-weighted magnetic resonance images and by staining brain sections with 2,3,5-triphenyltetrazolium chloride (TTC) 24 hours after MCAO.

Surgical induction of ischemic brain damage in the rat is a widely used model for stroke research. Here we demonstrate the induction of focal cerebral ischemia by occlusion of the middle cerebral artery. Visualization of the resulting infarct by histological staining and magnetic resonance imaging is also shown.

Stroke is the leading cause of disability and the third leading cause of death in adults worldwide1. In human stroke, there exists a highly variable ...

Electrophysiological Recordings from the Giant Fiber Pathway of D. melanogaster

When startled adult D. melanogaster react by jumping into the air and flying away. In many invertebrate species, including D. melanogaster, the "escape" (or "startle") response during the adult stage is mediated by the multi-component neuronal circuit called the Giant Fiber System (GFS). The comparative large size of the neurons, their distinctive morphology and simple connectivity make the GFS an attractive model system for studying neuronal circuitry. The GFS pathway is composed of two bilaterally symmetrical Giant Fiber (GF) interneurons whose axons descend from the brain along the midline into the thoracic ganglion via the cervical connective. In the mesothoracic neuromere (T2) of the ventral ganglia the GFs form electro-chemical synapses with 1) the large medial dendrite of the ipsilateral motorneuron (TTMn) which drives the tergotrochanteral muscle (TTM), the main extensor for the mesothoracic femur/leg, and 2) the contralateral peripherally synapsing interneuron (PSI) which in turn forms chemical (cholinergic) synapses with the motorneurons (DLMns) of the dorsal longitudinal muscles (DLMs), the wing depressors. The neuronal pathway(s) to the dorsovental muscles (DVMs), the wing elevators, has not yet been worked out (the DLMs and DVMs are known jointly as indirect flight muscles - they are not attached directly to the wings, but rather move the wings indirectly by distorting the nearby thoracic cuticle) (King and Wyman, 1980; Allen et al., 2006). The di-synaptic activation of the DLMs (via PSI) causes a small but important delay in the timing of the contraction of these muscles relative to the monosynaptic activation of TTM (~0.5 ms) allowing the TTMs to first extend the femur and propel the fly off the ground. The TTMs simultaneously stretch-activate the DLMs which in turn mutually stretch-activate the DVMs for the duration of the flight. The GF pathway can be activated either indirectly by applying a sensory (e.g."air-puff" or "lights-off") stimulus, or directly by a supra-threshold electrical stimulus to the brain (described here). In both cases, an action potential reaches the TTMs and DLMs solely via the GFs, PSIs, and TTM/DLM motoneurons, although the TTMns and DLMns do have other, as yet unidentified, sensory inputs. Measuring "latency response" (the time between the stimulation and muscle depolarization) and the "following to high frequency stimulation" (the number of successful responses to a certain number of high frequency stimuli) provides a way to reproducibly and quantitatively assess the functional status of the GFS components, including both central synapses (GF-TTMn, GF-PSI, PSI-DLMn) and the chemical (glutamatergic) neuromuscular junctions (TTMn-TTM and DLMn-DLM). It has been used to identify genes involved in central synapse formation and to assess CNS function.

Electrophysiological Recordings from the Giant Fiber Pathway of D. melanogaster

The Giant Fiber System is a simple neuronal circuit of adult Drosophila melanogaster containing the largest neurons in the fly. We describe the protocol for monitoring synaptic transmission through this pathway by recording post synaptic potentials in dorsal longitudinal (DLM) and tergotrochanteral (TTM) muscles following direct stimulation of the Giant Fiber interneurons.

When startled adult D. melanogaster react by jumping into the air and flying away. In many invertebrate species, including D. melanogaster, the "escape" ...

Whole Mount Preparation of the Adult Drosophila Ventral Nerve Cord for Giant Fiber Dye Injection

To analyze the axonal and dendritic morphology of neurons, it is essential to obtain accurate labeling of neuronal structures. Preparing well labeled samples with little to no tissue damage enables us to analyze cell morphology and to compare individual samples to each other, hence allowing the identification of mutant anomalies.
In the demonstrated dissection method the nervous system remains mostly inside the adult fly. Through a dorsal incision, the abdomen and thorax are opened and most of the internal organs are removed. Only the dorsal side of the ventral nerve cord (VNC) and the cervical connective 
(CvC) containing the big axons of the giant fibers (GFs)1 are exposed, while the brain containing the GF cell body and dendrites remains2 in the 
intact head. In this preparation most nerves of the VNC should remain attached to their muscles. 
Following the dissection, the intracellular filling of the giant fiber (GF) with a fluorescent dye is demonstrated. In the CvC the GF axons are located at the dorsal surface and thus can be easily visualized under a microscope with differential interference contrast (DIC) optics. This allows the injection of the GF axons with dye at this site to label the entire GF including the axons and their terminals in the VNC. This method results in reliable and strong staining of the GFs allowing the neurons to be imaged immediately after filling with an epifluorescent microscope. Alternatively, the fluorescent signal can be enhanced using standard immunohistochemistry procedures3 suitable for high resolution confocal microscopy.

Whole Mount Preparation of the Adult Drosophila Ventral Nerve Cord for Giant Fiber Dye Injection

An in vivo dissection of the adult Drosophila ventral nerve cord (VNC) is demonstrated. This particular dissection method causes little damage to the VNC allowing the subsequent labeling of the giant fiber neurons with fluorescent dye for high resolution imaging.

To analyze the axonal and dendritic morphology of neurons, it is essential to obtain accurate labeling of neuronal structures. Preparing well labeled ...

Evaluation of Bioenergetic Function in Cerebral Vascular Endothelial Cells

The integrity of the blood-brain-barrier (BBB) is critical to prevent brain injury. Cerebral vascular endothelial (CVE) cells are one of the cell types that comprise the BBB; these cells have a very high-energy demand, which requires optimal mitochondrial function. In the case of disease or injury, the mitochondrial function in these cells can be altered, resulting in disease or&#160;the opening of the BBB. In this manuscript, we introduce a method to measure mitochondrial function in CVE cells by using whole, intact cells and a bioanalyzer. A mito-stress assay is used to challenge the cells that have been perturbed, either physically or chemically, and evaluate their bioenergetic function. Additionally, this method also provides a useful way to screen new therapeutics that have direct effects on mitochondrial function. We have optimized the cell density necessary to yield oxygen consumption rates that allow for the calculation of a variety of mitochondrial parameters, including ATP production, maximal respiration, and spare capacity. We also show the sensitivity of the assay by demonstrating that the introduction of the&#160;microRNA, miR-34a, leads to a pronounced and detectable decrease in mitochondrial activity. While the data shown in this paper is optimized for the bEnd.3 cell line, we have also optimized the protocol for primary CVE cells, further suggesting the utility in preclinical and clinical models.

Endothelial cell mitochondria are critical to maintain blood-brain-barrier integrity. We introduce a protocol to measure bioenergetic function in cerebral vascular endothelial cells.

The integrity of the blood-brain-barrier (BBB) is critical to prevent brain injury. Cerebral vascular endothelial (CVE) cells are one of the cell types ...

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

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

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

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

Optimized Management of Endovascular Treatment for Acute Ischemic Stroke

This manuscript describes a streamlined protocol for the management of patients with acute ischemic stroke, which aims at the minimization of time from hospital admission to reperfusion. Rapid restoration of cerebral blood flow is essential for the outcomes of patients with acute ischemic stroke. Endovascular treatment (EVT) has become the standard of care to accomplish this in patients with acute stroke due to large vessel occlusion (LVO). To achieve reperfusion of ischemic brain regions as fast as possible, all in-hospital time delays have to be carefully avoided. Therefore, management of patients with acute ischemic stroke was optimized with an interdisciplinary standard operating procedure (SOP). Stroke neurologists, diagnostic as well as interventional neuroradiologists, and anesthesiologists streamlined all necessary processes from patient admission and diagnosis to EVT of eligible patients. Target times for every step were established. Actually achieved times were prospectively recorded along with clinical data and imaging scores for all endovascularly treated stroke patients. These data were regularly analyzed and discussed in interdisciplinary team meetings. Potential issues were evaluated and all staff involved was trained to adhere to the SOP. This streamlined patient management approach and enhanced interdisciplinary collaboration reduced time from patient admission to reperfusion significantly and was accompanied by a beneficial effect on clinical outcomes.

The outcome of patients with acute ischemic stroke depends on swift restoration of cerebral blood flow. This protocol aims at optimizing the management of such patients by minimizing peri-procedural timings and rendering the time from hospital admission to reperfusion as short as possible.

This manuscript describes a streamlined protocol for the management of patients with acute ischemic stroke, which aims at the minimization of time from ...

Data Acquisition and Analysis In Brainstem Evoked Response Audiometry In Mice

Brainstem evoked response audiometry (BERA) is of central relevance in the clinical neurophysiology. As other evoked potential (EP) techniques, such as visually evoked potentials (VEPs) or somatosensory evoked potentials (SEPs), the auditory evoked potentials (AEPs) are triggered by the repetitive presentation of identical stimuli, the electroencephalographic (EEG) response of which is subsequently averaged resulting in distinct positive (p) and negative (n) deflections. In humans, both the amplitude and the latency of individual peaks can be used to characterize alterations in synchronization and conduction velocity in the underlying neuronal circuitries. Importantly, AEPs are also applied in basic and preclinical science to identify and characterize the auditory function in pharmacological and genetic animal models. Even more, animal models in combination with pharmacological testing are utilized to investigate for potential benefits in the treatment of sensorineural hearing loss (e.g., age- or noise-induced hearing deficits). Here we provide a detailed and integrative description of how to record auditory brainstem-evoked responses (ABRs) in mice using click and tone-burst application. A specific focus of this protocol is on pre-experimental animal housing, anesthesia, ABR recording, ABR filtering processes, automated wavelet-based amplitude growth function analysis, and latency detection.

Brainstem evoked response audiometry is an important tool in clinical neurophysiology. Nowadays, brainstem evoked response audiometry is also applied in the basic science and preclinical studies involving both pharmacological and genetic animal models. Here we provide a detailed description of how auditory brainstem responses can be successfully recorded and analyzed in mice.

Brainstem evoked response audiometry (BERA) is of central relevance in the clinical neurophysiology. As other evoked potential (EP) techniques, such as ...

Simulator Training for Endovascular Neurosurgery

Simulation-based training has become common practice across medical specialties, especially for learning complex skills performed in high-risk environments. In the field of endovascular neurosurgery, the demand for consequence- and risk-free learning environments led to the development of simulation devices valuable for medical trainees. The goal of this protocol is to provide instructive guidelines for the use of an endovascular neurosurgery simulator in an academic setting. The simulator provides trainees with the opportunity to receive realistic feedback on their knowledge of anatomy, as well as haptic feedback indicative of their success in handling the catheter-based systems without negative consequences. The utility of this specific protocol in relation to other neuroendovascular training modalities is also discussed.

Simulation of complex, high-risk procedures is critical to the education of medical trainees. A protocol for simulator-based endovascular neurosurgery training in a controlled academic environment is described. The protocol includes stepwise guidelines for trainees of varying levels, with a discussion of the advantages and limitations of this model.

Simulation-based training has become common practice across medical specialties, especially for learning complex skills performed in high-risk ...

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 ...