This study was supported by the 'Medical Scientific Fund of the Major of the City of Vienna' and Ethicon Biosurgery, Inc. New Jersey, USA. The authors express their thanks to Professor Heber Ferraz-Leite, Director of the 'European Workshop on Microsurgery and Cerebral Revascularization' at the Medical University of Vienna, Austria, for his valuable microsurgical teaching.

1. Positioning and preparation of the animal
The experiments were approved by the responsible local ethical committee and performed according to the Felasa guidelines.
<ol>
	<li>Start general anesthesia of the female New Zealand White rabbit (2.5-3.5 kg) with intramuscular injection of Ketamin (30mg/kg) and Xylazine (6mg/kg) and continue after mechanical intubation (tube diameter 4mm, length 18mm) with gas anesthesia (Isoflurane) under ECG monitoring. Perform pain monitoring via toe pinch all 15 minutes and adapt anesthesia in case of positive response. </li>
 <li>Fix the rabbit in supine position on a bodywarm plate.</li>
 <li>Prepare the area for surgery by removal of hair reaching from the angle of jaw down to the middle of the thorax. Perform all procedures under sterile conditions. </li>
</ol>
2. Preparation of the external jugular vein
<ol>
 <li>Perform a midline incision from the angle of jaw down to the manubrium sterni. </li>
 <li>Using the operation microscope, select a 1 cm long segment of the left external jugular vein without venous branches and isolate it microsurgically (Figure 1a). During the whole operation frequently apply 4% papaverin HCl solution and antibiotic solution (neomycin sulfate 5mg/ml) topically on the vessels and anastomoses to prevent vasospasm and local infections.</li>
 <li>Ligate the vein proximally and distally with 4-0 polyfilament sutures (Vicryl, Ethicon Inc, New Jersey, USA), then resect it and keep it in heparinized saline (1000 IU heparin in 20 ml 0.9% saline and 1 ml 4% papaverin HCl ). </li>
</ol>
3. Preparation of both common carotid arteries (CCAs)
<ol>
	<li>Prepare a segment of the left CCA reaching from the carotid bifurcation down to the aortic arch. Preserve as far as possible arterial branches running medially and supplying laryngeal and tracheal structures as well as all neural structures. </li>
 <li>Prepare the right CCA. Mobilize it up to the carotid bifurcation and down to the bracchiocephalic trunk. </li>
 <li>Administer 1000 IU heparin intravenously. </li>
 <li>Clip the right CCA temporarily just below the carotid bifurcation.</li>
 <li>Ligate the right CCA proximally directly above the brachiocephalic trunk. Cut the right CCA above the ligature and irrigate the stump with heparinized saline. </li>
 <li>Cut and resect carefully the adventitia at the free end of the right CCA. Free meticulously the segment of the left CCA planned for the anastomosis of the adventitia and clip it distally and proximally. </li>
</ol>
4. Anastomosis of both CCAs
<ol>
	<li> Perform an elliptical arteriotomy of the left CCA between the clips according to the size of the planned anastomosis with the right CCA and the venous pouch. Irrigate the left CCA with heparinized saline. (Figure 1b)</li>
 	<li> Suture the posterior circumference of the right CCA-stump into the arteriotomy of the left CCA, using four to five non-resorbable 10-0 monofilament sutures (Ethilon 10-0, Ethicon Inc, New Jersey, USA). </li>
 <li>Make a longitudinal cut in the stump of the right CCA to adapt to half the circumference of the venous pouch. Anastomose the back side of the venous pouch wall first with the arteriotomy in the left CCA, using again 4 to 5 10-0 sutures. Then anastomose the back side of the venous pouch with the backside of the right CCA with 3 to 4 sutures. (Figure 1c)</li>
 <li>Perform the anastomosis in the same order at the anterior side. (Figure 1d)</li>
 <li>Remove the distal clip on the right CCA. The aneurysm is usually not completely sealed, therefore wash trapped air and debris out. </li>
 <li>Seal the suture lines around the anastomosis and the aneurysm neck with a fat pad and fibrin glue (Evicel, Ethicon Biosurgery Inc., New Jersey, USA). Remove the remaining clips.</li>
 <li>Use 4-0 resorbable sutures (Monocryl,Ethicon Inc, New Jersey, USA) for interrupted subcutaneous sutures and then for a running cutaneus suture.</li>
</ol>
5. Postoperative management
<ol>
	<li>Administer 10 mg/kg acetylsalicylic acid intravenously, and 60 ml 5% glucose subcutaneously.</li>
	<li>Apply transdermal fentanyl matrix patches releasing 12.5 &mu;g/hour in the shaved neck region of the animals for effective analgesia during 72 hours. </li>
	<li>Administer 250 IU/kg low molecular heparin daily subcutaneously for 2 weeks. </li>
</ol>
6. Representative Results: 
If the procedure is done correctly, the aneurysm will stay patent and the animals will recover within about 3 days. In our experience an imaging procedures should be done at minimum one week later. Otherwise the animals possibly won&#x2019;t tolerate the procedure and could be lost. For embolization we would recommend to wait for about 4 weeks to guarantee complete endothelialization of the aneurysm neck.Using the presented techniques and management the authors could reach aneurysm patency in 85.7% and no mortality in their recently published series.14 To minimize the high rate of reported intestinal complications of up to 20%,12 we provided a prolonged postoperative analgetic management of 72 hour analgesia as compared to routine 24 hour analgesia.This intensified management resulted in no gastric complications. The aggressive anticoagulation management is important to reach sufficient aneurysm patency rates. This goes along with the findings of Grunwald et al. showing the positive effects of combined anticoagulation.17 Because of the aggressive anticoagulation it is crucial to have an effective sealing of the anastomosis. We made very good experience using new fibrin glue (Evicel, Ethicon Biosurgery Inc., New Jersey, USA). Additionally we recommend using small rabbits (maximum 3.5 kg), as in our experience complications are often related to overweight subjects.

<ol>
	<li>Molyneux, A., Kerr, R., Yu, L. <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, 809-817 (2005).</li><li>Mitchell, P., Kerr, R., Mendelow, A., Molyneux, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Could+late+rebleeding+overturn+the+superiority+of+cranial+aneurysm+coil+embolization+over+clip+ligation+seen+in+the+International+Subarachnoid+Aneurysm+Trial.">Could late rebleeding overturn the superiority of cranial aneurysm coil embolization over clip ligation seen in the International Subarachnoid Aneurysm Trial.</a> J Neurosur. 108, 437-442 (2008).</li><li>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=Histopathological+findings+in+experimental+aneurysms+embolized+with+conventional+and+thrombogenic/antithrombolytic+Guglielmi+coils.">Histopathological findings in experimental aneurysms embolized with conventional and thrombogenic/antithrombolytic Guglielmi coils.</a> Minim Invasive Neurosurg. 42, 167-174 (1999).</li><li>Bocher-Schwarz, H. G. <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, 379-384 (2002).</li><li>Sherif, C., Plenk, H., 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, 559-566 (2006).</li><li>Massoud, T., Guglielmi, G., Ji, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experiemental+saccular+aneurysms.+1.+Review+of+surgically+constructed+models+and+their+laboratory+applications.">Experiemental saccular aneurysms. 1. Review of surgically constructed models and their laboratory applications.</a> Neurorad. 36, 537-546 (1994).</li><li>Anidjar, S., Salzmann, J., Gentric, D. <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, 973-981 (1990).</li><li>Wakhloo, A., Schellhammer, F., de Vries, J. <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. 15, 493-502 (1994).</li><li>Dai, D. <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> AJNR Am J Neuroradiol. 26, 2560-2568 (2005).</li><li>Shin, Y. S. <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> AJNR. 26, 1764-1767 (2005).</li><li>Abruzzo, T. <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 Am J Neuroradiol. 19, 1309-1314 (1998).</li><li>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=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> Minim Invasive Neurosurg. 41, 129-132 (1998).</li><li>Spetzger, U. <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> J Neurosurg. 85, 488-495 (1996).</li><li>Sherif, C., Marbacher, S., Erhardt, S., Fandino, J., 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> AJNR. , .</li><li>Marbacher, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complex+Bi-Lobular,+Bi-Saccular,+and+Broad-Necked+Microsurgical+Aneurysms+Formation+in+the+Rabbit+Bifurcation+Model+for+the+Study+of+Upcoming+Endovascular+Techniques.">Complex Bi-Lobular, Bi-Saccular, and Broad-Necked Microsurgical Aneurysms Formation in the Rabbit Bifurcation Model for the Study of Upcoming Endovascular Techniques.</a> AJNR. , .</li><li>Sherif, C., Plenk, H. <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> AJNR. , .</li><li>Grunwald, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anticoagulation+regimes+and+their+influence+on+the+occlusion+rate+of+aneurysms:+an+experimental+study+in+rabbits.">Anticoagulation regimes and their influence on the occlusion rate of aneurysms: an experimental study in rabbits.</a> Neurosurg. 57, 1048-1054 (2005).</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> AJNR Am J Neuroradiol. 10, 400-402 (1989).</li></ol>

No conflicts of interest declared.

The authors recommend to considerate the following critical surgical key steps to achieve good results and patent aneurysms:
<ol>
 <li>Careful dissection of a long segment of the left CCA for easy anastomosis without destruction of nerves and small vessels.</li>
 <li>Careful removal of the highly thrombogenic periadventitial fibrous tissue prior to the creation of the anastomosis.</li>
 <li>Creation of a tensionless anastomosis. </li>
 <li>Fewer sutures: The proposed sequence of anastomsis sites beginning at the back...

<table border="1">
	<tbody>
		<tr>
			<th>Name of the reagent</th>
			<th>Company</th>
			<th>Catalogue number</th>
			<th>Comments</th>
		</tr>
		<tr>
			<td>Vicryl4-0 polyfilament restorable sutures </td>
			<td>Ethicon Inc</td>
			<td>J386H</td>			
			<td>&nbsp;</td>			
		</tr>
		<tr>
			<td>Ethilon 10-0 monofil non resorbable sutures </td>
			<td>Ethicon Inc</td>
			<td>2814</td>
			<td> taper point needle</td>
		</tr>
		<tr>			
			<td>Evicel Bioglue </td>
			<td>Ethicon Biosurgery Inc.</td>
 			<td>3901</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Fentanyl dermal patch 12.5 &mu;g/h</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Any genericon</td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Any genericon</td>
		</tr>
		<tr>
			<td>0.9% Saline</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Any genericon</td>
		</tr>			
		<tr>
			<td>4% Papaverin HCl</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Any genericon</td>
		</tr>
		<tr>
			<td>Neomycin sulfate 5 mg/ml</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Any genericon</td>
		</tr>
		<tr>
			<td>Ketamin 50mg/ml</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Any genericon</td>
		</tr>
 		<tr>
 			<td>Xylazine 20mg/ml</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Any genericon</td>
		</tr>
	</tbody>
</table>

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.

Watch this Scientific Journal Video about Microsurgical Venous Pouch Arterial-Bifurcation Aneurysms in the Rabbit Model: Technical Aspects at JoVE.com

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

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 ...

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13.9K Views.  Hospital Rudolfstiftung. 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.

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

Technical Aspects of the Mouse Aortocaval Fistula

Technical aspects of creating an arteriovenous fistula in the mouse are discussed. Under general anesthesia, an abdominal incision is made, and the aorta and inferior vena cava (IVC) are exposed. The proximal infrarenal aorta and the distal aorta are dissected for clamp placement and needle puncture, respectively. Special attention is paid to avoid dissection between the aorta and the IVC. After clamping the aorta, a 25 G needle is used to puncture both walls of the aorta into the IVC. The surrounding connective tissue is used for hemostatic compression. Successful creation of the AVF will show pulsatile arterial blood flow in the IVC. Further confirmation of successful AVF can be achieved by post-operative Doppler ultrasound.

The goal is to produce an arteriovenous fistula that is simple and reproducible. This method does not use sutures or glue adhesive. Therefore the samples can be used with the least amount of foreign materials for analysis.

Technical aspects of creating an arteriovenous fistula in the mouse are discussed. Under general anesthesia, an abdominal incision is made, and the aorta ...

The Helsinki Rat Microsurgical Sidewall Aneurysm Model

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

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

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

The Rabbit Blood-shunt Model for the Study of Acute and Late Sequelae of Subarachnoid Hemorrhage: Technical Aspects

Early brain injury and delayed cerebral vasospasm both contribute to unfavorable outcomes after subarachnoid hemorrhage (SAH). Reproducible and controllable animal models that simulate both conditions are presently uncommon. Therefore, new models are needed in order to mimic human pathophysiological conditions resulting from SAH.
This report describes the technical nuances of a rabbit blood-shunt SAH model that enables control of intracerebral pressure (ICP). An extracorporeal shunt is placed between the arterial system and the subarachnoid space, which enables examiner-independent SAH in a closed cranium. Step-by-step procedural instructions and necessary equipment are described, as well as technical considerations to produce the model with minimal mortality and morbidity. Important details required for successful surgical creation of this robust, simple and consistent ICP-controlled SAH rabbit model are described.

The experimental intracranial pressure-controlled blood shunt subarachnoid hemorrhage (SAH) model in the rabbit combines the standard procedures &#8212; subclavian artery cannulation and transcutaneous cisterna magna puncture, which enables close mimicking of human pathophysiological conditions after SAH. We present step-by-step instructions and discuss key surgical points for successful experimental SAH creation.

Early brain injury and delayed cerebral vasospasm both contribute to unfavorable outcomes after subarachnoid hemorrhage (SAH). Reproducible and ...

A Murine Model of Arterial Restenosis: Technical Aspects of Femoral Wire Injury

Cardiovascular disease caused by atherosclerosis is the leading cause of death in the developed world. Narrowing of the vessel lumen, due to atherosclerotic plaque development or the rupturing of established plaques, interrupts normal blood flow leading to various morbidities such as myocardial infarction and stroke. In the clinic endovascular procedures such as angioplasty are commonly performed to reopen the lumen. However, these treatments inevitably damage the vessel wall as well as the vascular endothelium, triggering an excessive healing response and the development of a neointimal plaque that extends into the lumen causing vessel restenosis (re-narrowing). Restenosis remains a major cause of failure of endovascular treatments for atherosclerosis. Thus, preclinical animal models of restenosis are vitally important for investigating the pathophysiological mechanisms as well as translational approaches to vascular interventions. Among several murine experimental models, femoral artery wire injury is widely accepted as the most suitable for studies of post-angioplasty restenosis because it closely resembles the angioplasty procedure that injures both endothelium and vessel wall. However, many researchers have difficulty utilizing this model due to its high degree of technical difficulty. This is primarily because a metal wire needs to be inserted into the femoral artery, which is approximately three times thinner than the wire, to generate sufficient injury to induce prominent neointima. Here, we describe the essential surgical details to effectively overcome the major technical difficulties of this model. By following the presented procedures, performing the mouse femoral artery wire injury becomes easier. Once familiarized, the whole procedure can be completed within 20 min.

The mouse femoral artery wire injury model of restenosis is technically challenging. In this protocol we show the key technical details essential for successfully performing wire injury to induce consistent&#160;neointima for studies of restenosis.

Cardiovascular disease caused by atherosclerosis is the leading cause of death in the developed world. Narrowing of the vessel lumen, due to ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

Transthoracic Echocardiographic Examination in the Rabbit Model

Large animal models such as the rabbit are valuable for translational preclinical research. Rabbits have a similar cardiac electrophysiology compared to that of humans and that of other large animal models such as dogs and pigs. However, the rabbit model has the additional advantage of lower maintenance costs compared to other large animal models. The longitudinal evaluation of cardiac function using echocardiography, when appropriately implemented, is a useful methodology for preclinical assessment of novel therapies for heart failure with reduced ejection fraction (e.g. cardiac regeneration). The correct use of this non-invasive tool requires the implementation of a standardized examination protocol following international guidelines. Here we describe, step by step, a detailed protocol supervised by veterinary cardiologists for performing echocardiography in the rabbit model, and demonstrate how to correctly obtain the different echocardiographic views and imaging planes, as well as the different imaging modes available in a clinical echocardiography system routinely used in human and veterinary patients.

Here we describe, step by step, a detailed protocol for performing echocardiography in the rabbit model. We show how to correctly obtain the different echocardiographic views and imaging planes, as well as the different imaging modes available in a clinical echocardiography system routinely used in human and veterinary patients.

Large animal models such as the rabbit are valuable for translational preclinical research. Rabbits have a similar cardiac electrophysiology compared to ...

A Rabbit Venous Interposition Model Mimicking Revascularization Surgery using Vein Grafts to Assess Intimal Hyperplasia under Arterial Blood Pressure

Although vein grafts have been commonly used as autologous grafts in revascularization surgeries for ischemic diseases, the long-term patency remains poor because of the acceleration of intimal hyperplasia due to the exposure to arterial blood pressure. The present protocol is designed for the establishment of experimental venous intimal hyperplasia by interposing rabbit jugular veins to the ipsilateral carotid arteries. The protocol does not require surgical procedures deep in the body trunk and the extent of the incision is limited, which is less invasive for the animals, allowing long-term observation after implantation. This simple procedure enables researchers to investigate strategies to attenuate the progression of intimal hyperplasia of the implanted vein grafts. Using this protocol, we reported the effects transduction of microRNA-145 (miR-145), which is known to control the phenotype of vascular smooth muscle cells (VSMCs) from the proliferative to the contractile state, into harvested vein grafts. We confirmed the attenuation of intimal hyperplasia of vein grafts by transducing miR-145 before implantation surgery through the phenotype change of the VSMCs. Here we report a less invasive experimental platform to investigate the strategies that can be used to attenuate intimal hyperplasia of vein grafts in revascularization surgeries.

The present protocol aims to experimentally create venous intimal hyperplasia by subjecting veins to arterial blood pressure for developing strategies to attenuate venous intimal hyperplasia following revascularization surgery using vein grafts.

Although vein grafts have been commonly used as autologous grafts in revascularization surgeries for ischemic diseases, the long-term patency remains poor ...

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

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

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

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

Isolated Lung Perfusion System in the Rabbit Model

The isolated lung perfusion system has been widely used in pulmonary research, contributing to elucidate the lungs&#39; inner workings, both micro and macroscopically. This technique is useful in the characterization of pulmonary physiology and pathology by measuring metabolic activities and respiratory functions, including interactions between circulatory substances and the effects of inhaled or perfused substances, as in drug testing. While in vitro methods involve the slicing and culturing of tissues, the isolated ex vivo lung perfusion system allows to work with a complete functional organ making possible the study of a continuous physiological function while recreating ventilation and perfusion. However, it should be noted that the effects of the absence of central innervation and lymphatic drainage still have to be fully assessed. This protocol aims to describe the assembly of the isolated lung apparatus, followed by the surgical extraction and cannulation of lungs and heart from experimental lab animals, as well as to display the perfusion technique and signal processing of data. The average viability of the isolated lung ranges between 5-8 h; during this period, the pulmonary capillary permeability increases, causing edema and lung injury. The functionality of preserved pulmonary tissue is measured by the capillary filtration coefficient (Kfc), used to determine the extent of pulmonary edema through time.

The isolated rabbit lung preparation is a gold standard tool in pulmonary research. This publication aims to describe the technique as developed for the study of physiological and pathological mechanisms involved in airway reactivity, lung preservation, and preclinical research in lung transplantation and pulmonary edema.

The isolated lung perfusion system has been widely used in pulmonary research, contributing to elucidate the lungs&#39; inner workings, both micro and ...

A Rabbit Aortic Valve Stenosis Model Induced by Direct Balloon Injury

Animal models are emerging as an important tool to understand the pathologic mechanisms underlying aortic valve stenosis (AVS) because of the lack of access to reliable sources of diseased human aortic valves. Among the various animal models, AVS rabbit models are one of the most commonly used in large animal studies. However, traditional AVS rabbit models require a long-term period of dietary supplementation and genetic manipulation to induce significant stenosis in the aortic valve, limiting their use in experimental studies. To address these limitations, a new AVS rabbit model is proposed, in which stenosis is induced by a direct balloon injury to the aortic valve. The present protocol describes a successful technique for inducing AVS in New Zealand white (NZW) rabbits, with step-by-step procedures for the preparation, the surgical procedure, and the post-operative care. This simple and reproducible model offers a promising approach for studying the initiation and progression of AVS and provides a valuable tool for investigating the underlying pathological mechanisms of the disease.

An appropriate animal model is needed to understand the pathologic mechanisms underlying aortic valve stenosis (AVS) and to evaluate the efficacy of therapeutic interventions. The present protocol describes a new procedure for developing the AVS rabbit model via a direct balloon injury in vivo.

Animal models are emerging as an important tool to understand the pathologic mechanisms underlying aortic valve stenosis (AVS) because of the lack of ...