This work was supported by Grants-in-Aid for Scientific Research (B) (20291958) to N.U.; Grants-in-Aid for Young Scientists (A) (25713024) to N.Z.

Animal care and experiments were performed in accordance with the guidelines of the Hamamatsu University School of Medicine Animal Care Committee at the Center for Animal Care.
1. Surgical Procedure for Creating the Model
NOTE: Place the surgical instruments into a bead sterilizer for 10 s preoperatively. Use sterile gloves intraoperatively.
<ol>
	<li>Use male rats weighing 300 - 400 g. Allow the rats access to food and water ad libitum.</li>
	<li>Anesthetize the rat with isoflurane inhalation (2.0 - 3.0 mL/L). Confirm proper anesthetization by toe pinch.</li>
	<li>Shave hair on the abdomen with an electric razor, and scrub the abdomen with alcohol and a povidone-iodine solution.</li>
	<li>Place the rat in the supine position on the operation table. Use vet ointment on the rat&#39;s eyes to prevent dryness while under anesthesia.</li>
	<li>Perform a laparotomy with a ventral midline abdominal incision using a scissors. To secure a clear surgical field, pack the abdominal contents inside the abdominal cavity with sterile gauze using a wound retractor.</li>
	<li>To detach the aorta from the perivascular tissue, gently pick up and tear the retroperitoneum with tweezers to expose the aortic wall and exfoliate the infrarenal aorta in the retroperitoneal space from the level of the left renal vein to the bifurcation from the perivascular tissue (Figure 1A).</li>
	<li>Ligate the vessels branching from the abdominal aorta with a 5-0 silk string to block blood supply at a point away from the aorta that does not narrow the aortic lumen.</li>
	<li>To block aortic blood flow, place vascular clips just below the renal artery and just above the bifurcation of the aorta. 
	NOTE: The size of the clips should be larger than the aortic diameter to block the blood flow completely. A temporary 5-0 silk string ligature is also sufficient for blocking blood flow instead of the clips.</li>
	<li>Similar to creating a dot hemorrhage on the surface of the aortic wall, cut the anterior aortic wall using a micro-scissor&#160;5 mm distally from the aortic clip, adjacent to the renal artery branches, to insert a polyurethane catheter.</li>
	<li>Insert a polyurethane catheter (outside diameter 0.55 mm, inside diameter 0.37 mm) using a 24-gauge indwelling needle shallowly through the incision to wash away blood in the aorta with water. Administer a 1-mL syringe filled with water into the polyurethane catheter, and wash away the remaining blood in the aorta with water. After irrigation, remove the catheter from the aorta.</li>
	<li>Before inserting a polyurethane catheter (outside diameter 1.20 mm, inside diameter 0.94 mm) using an 18-gauge indwelling needle, cut the polyurethane catheter to 10 mm long (Figure 1B).
	<ol>
		<li>Insert the cut piece of the polyurethane catheter into the aortic lumen from the incision made in step 1.9. Completely place the 10-mm catheter into the aortic lumen, and reposition the midpoint of the catheter to match the location of the incision (i.e., cover the incision with the catheter).</li>
	</ol>
	</li>
	<li>Repair the incision with interrupted&#160;sutures using an 8-0 monofilament string (Figure 1C).</li>
	<li>Ligate the abdominal aorta using a combination of 5-0 silk string and the polyurethane catheter (Figure 1D). 
	NOTE: The aortic lumen can be maintained by the indwelling catheter. Ligate the aorta firmly to prevent misaligning the catheter position. The surgeon&#39;s knot is recommended, because it adds an extra twist when tying the first throw of the knot, forming a double overhand knot, and an additional turn provides more friction and can reduce loosening.</li>
	<li>After ligation, remove the vascular clip at the aortic bifurcation, and then, remove the vascular clip below the renal artery to restore the antegrade blood flow. Confirm gross aortic pulsation.</li>
	<li>Close the abdominal incision in two layers, the peritoneum and other layers, with a 4-0 polypropylene suture. Tightly suture the incision to prevent a protruded organ.</li>
	<li>Apply topical lidocaine on the abdominal incision. Place the rat on a heating pad until consciousness returns. Do not leave a rat unattended until it has regained sufficient consciousness to maintain sternal recumbency. Do not return a rat that has undergone operation to the company of other rats until it has fully recovered.</li>
	<li>Monitor the rat closely after the operation. Inject a bolus of sterile saline (1.0 - 2.0 mL) subcutaneously if a rat shows signs of distress or body weight loss.</li>
	<li>Postoperatively, observe the time-course development of the enlarged aorta on an ultrasound echogram. Measure the maximum diameter from the inside edge to the outside edge of the abdominal aorta as described5.</li>
</ol>
2. Harvesting, Fixing, and Elastica-van Gieson (EVG) Staining
<ol>
	<li>Twenty-eight days postoperatively, administer sodium pentobarbital &#8805; 100 mg/kg intraperitoneally to euthanize the rats.</li>
	<li>Cut open the rat&#39;s abdominal cavities ventrally. Make the initial incision using a scalpel. Harvest the aneurysm using scissors and place the harvested aortas in 10% neutrally buffered formalin for 24 - 48 h. 
	NOTE: The surgeon must be careful to prevent injury to the aorta and other organs when exposing the aneurysm. An elevated abdominal organ should be observed, indicating AAA formation. Figure 2 shows an in vivo image and ex vivo image; this representative sample of AAAs is not ruptured.</li>
	<li>EVG staining9
	<ol>
		<li>Solution Preparation
		<ol>
			<li>Prepare Verhoeff&#39;s hematoxylin by mixing the following in the given order: alcoholic hematoxylin 20 mL, 10% ferric chloride 8.0 mL, and Lugol&#39;s iodine 8 mL. Mix the solution between each addition. This solution can be prepared fresh as needed.</li>
			<li>Prepare Van Gieson&#39;s solution by mixing 1% acid fuchsin 1 mL and saturated picric acid 45 mL. Allow to stand overnight and mix well. This solution is stable for 2 weeks.</li>
			<li>Prepare differentiating solution (2% ferric chloride) by mixing 10% FeCl3 10 mL in 40 mL distilled water. This solution may be prepared fresh as needed.</li>
			<li>Prepare 5% hypo by dissolving 5.0 g sodium thiosulfate in 100 mL distilled water. This solution is stable for 1 year.</li>
		</ol>
		</li>
		<li>Staining procedure 
		NOTE: See reference9 for details.
		<ol>
			<li>Briefly, hydrate the fixed paraffin sections of the harvested aortas in distilled water for 10 min. Place the aortas in Verhoeff&#39;s hematoxylin for 25 min. Wash with water and dip 10-30 times in differentiating solution (2% FeCl3). Rinse in water. Place in 5% hypo for 1 min. Wash in water. Place in Van Gieson&#39;s solution for 5 min.</li>
			<li>Dehydrate in alcohol in the following order: 75%, 90%, 95%, 100%, and 100% alcohol for 30 s each. Then, clear in xylene for 5 min, twice. Place on a coverslip with resinous mounting medium. 
			NOTE: Elastic fibers appear blue-black to black; nuclei: blue to black; collagen: red; and other tissue elements: yellow.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

<ol><li>Wolinsky, H., Glagov, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Comparison+of+abdominal+and+thoracic+aortic+medial+structure+in+mammals.+Deviation+of+man+from+the+usual+pattern%5BTitle%5D)+AND+%22Circ+Res%22%5BJournal%5D)">Comparison of abdominal and thoracic aortic medial structure in mammals. Deviation of man from the usual pattern.</a> Circ Res. 25 (6), 677-686 (1969).</li>
<li>Sano, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Lymphangiogenesis+and+angiogenesis+in+abdominal+aortic+aneurysm%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Lymphangiogenesis and angiogenesis in abdominal aortic aneurysm.</a> PLoS One. 9 (3), e89830(2014).</li>
<li>Vorp, D. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Association+of+intraluminal+thrombus+in+abdominal+aortic+aneurysm+with+local+hypoxia+and+wall+weakening%5BTitle%5D)+AND+%22J+Vasc+Surg%22%5BJournal%5D)">Association of intraluminal thrombus in abdominal aortic aneurysm with local hypoxia and wall weakening.</a> J Vasc Surg. 34 (2), 291-299 (2001).</li>
<li>Tanaka, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Adventitial+vasa+vasorum+arteriosclerosis+in+abdominal+aortic+aneurysm%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Adventitial vasa vasorum arteriosclerosis in abdominal aortic aneurysm.</a> PLoS One. 8 (2), e57398(2013).</li>
<li>Tanaka, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Hypoperfusion+of+the+Adventitial+Vasa+Vasorum+Develops+an+Abdominal+Aortic+Aneurysm%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Hypoperfusion of the Adventitial Vasa Vasorum Develops an Abdominal Aortic Aneurysm.</a> PLoS One. 10 (8), e0134386(2015).</li>
<li>Tanaka, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Imaging+Mass+Spectrometry+Reveals+a+Unique+Distribution+of+Triglycerides+in+the+Abdominal+Aortic+Aneurysmal+Wall%5BTitle%5D)+AND+%22J+Vasc+Res%22%5BJournal%5D)">Imaging Mass Spectrometry Reveals a Unique Distribution of Triglycerides in the Abdominal Aortic Aneurysmal Wall.</a> J Vasc Res. 52 (2), 127-135 (2015).</li>
<li>Kugo, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+preventive+effect+of+fish+oil+on+abdominal+aortic+aneurysm+development%5BTitle%5D)+AND+%22Biosci+Biotechnol+Biochem%22%5BJournal%5D)">The preventive effect of fish oil on abdominal aortic aneurysm development.</a> Biosci Biotechnol Biochem. 80 (6), 1186-1191 (2016).</li>
<li>Kugo, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Adipocyte+in+vascular+wall+can+induce+the+rupture+of+abdominal+aortic+aneurysm%5BTitle%5D)+AND+%22Sci+Rep%22%5BJournal%5D)">Adipocyte in vascular wall can induce the rupture of abdominal aortic aneurysm.</a> Sci Rep. 6, 31268(2016).</li>
<li>Lichtenberg, A., Cebotari, S., Tudorache, I., Hilfiker, A., Haverich, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Biological+scaffolds+for+heart+valve+tissue+engineering%5BTitle%5D)+AND+%22Methods+Mol+Med%22%5BJournal%5D)">Biological scaffolds for heart valve tissue engineering.</a> Methods Mol Med. 140, 309-317 (2007).</li>
<li>Heistad, D. D., Marcus, M. L., Larsen, G. E., Armstrong, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Role+of+vasa+vasorum+in+nourishment+of+the+aortic+wall%5BTitle%5D)+AND+%22Am+J+Physiol%22%5BJournal%5D)">Role of vasa vasorum in nourishment of the aortic wall.</a> Am J Physiol. 240 (5), H781-H787 (1981).</li>
<li>Yoshimura, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Regression+of+abdominal+aortic+aneurysm+by+inhibition+of+c-Jun+N-terminal+kinase%5BTitle%5D)+AND+%22Nat+Med%22%5BJournal%5D)">Regression of abdominal aortic aneurysm by inhibition of c-Jun N-terminal kinase.</a> Nat Med. 11 (12), 1330-1338 (2005).</li>
<li>Azuma, J., Asagami, T., Dalman, R., Tsao, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Creation+of+murine+experimental+abdominal+aortic+aneurysms+with+elastase%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Creation of murine experimental abdominal aortic aneurysms with elastase.</a> J Vis Exp. (29), (2009).</li>
<li>Lu, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Subcutaneous+Angiotensin+II+Infusion+using+Osmotic+Pumps+Induces+Aortic+Aneurysms+in+Mice%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Subcutaneous Angiotensin II Infusion using Osmotic Pumps Induces Aortic Aneurysms in Mice.</a> J Vis Exp. (103), (2015).</li>
<li>United Kingdom, E. T. I., et al. <a target="_blank" href="http://www.nejm.org/doi/full/10.1056/NEJMoa0909305#t=article">Endovascular versus open repair of abdominal aortic aneurysm.</a> N Engl J Med. 362 (20), 1863-1871 (2010).</li>
</ol>

The authors have nothing to disclose.

Under physiological conditions, the inner layers of the aortic wall are nourished by diffusion from the luminal blood flow, whereas the outer and middle layers are nourished by the VV, which penetrate from the adventitia into the medial VV1. VV blood flow into the abdominal aortic wall can originate from the following three directions/areas: (1) the proximal direction through the aortic wall, (2) distal direction through the aortic wall, and (3) perivascular tissues10. Previously, our histological analysis of human tissues identified significant stenosis or occlusion of the VV in the AAA wall, suggesting that VV blood flow into the abdominal aortic wall can be reduced4. It is an extremely important point in this protocol that an infrarenal AAA was caused by a combination of polyurethane catheter insertion and suture ligation of the infrarenal abdominal aorta. To carefully exfoliate the tissue layer, surgeons must smoothly insert a polyurethane catheter into the aorta and firmly ligate the aorta to cause chronic hypoxia due to hypoperfusion of the adventitial VV and aneurysm formation. Using these techniques, blood flow in the aortic wall is consequently decreased, and a local hypoxic environment is induced. The blood flow reduction and hypoxia-induced aneurysm formation indicates that VV blood flow into the abdominal aortic wall plays a role in the pathogenesis of AAA formation.
Specifically, an aortic aneurysm model must satisfy the following conditions: a 1.5-fold increase in the vascular diameter compared to baseline, degeneration of the tunica media, and inflammation of the aortic wall. The most popular animal models have been constructed by inducing inflammatory responses using substances, such as CaCl211, elastase12, and angiotensin II13. These models can have a high reproducibility and obviously cause pathological change, and they have been commonly used in research studies. In our model, we assessed the aortic diameter using ultrasonography every 7 days from before the procedure was performed until day 28 after the procedure (Figure 3). Results showed that the aortic diameter moderately increased over the 28 days, indicating that this change in the diameter is similar to that in previous rodent models. Gross observation of the vascular form indicated a smooth fusiform shape (Figure 2). On day 28, we sacrificed the rats and performed histopathological analysis of the aortic tissue that was recovered. Tearing and disappearance of the elastic and collagen fibers of the tunica media and adventitia were observed (Figure 4). Moreover, inflammatory cells, such as macrophages, were present from the tunica adventitia to the tunica media.
Currently, the treatment options available for AAAs are limited to surgical repair and endovascular stent grafting, with mortality rates of 30 - 50% in patients with AAA rupture14. However, no drug has been approved for clinical use to treat AAAs. There is debate that there are discrepancies in the pathological findings between humans and established animal models used in AAA research. Similarities in the pathogenesis between human AAA and animal AAA models are essential for the development of pharmacological treatments. Regarding the effectiveness of rodent models, our rat model is morphologically similar to humans in terms of intraluminal thrombus5 and adipogenesis8. Furthermore, about 20% of the rats in this study had AAA rupture and died within 28 days after the procedure. Although aortic aneurysm rupture is the most critical event for this disease, rupture is uncommon with established experimental AAA models, and the mechanism has not yet been elucidated. Therefore, this model is useful for understanding the mechanism of dilation of the aortic diameter and rupture of the aneurysm.
The creation of this model is required for some surgical procedures. Therefore, researchers must practice creating this model, which is a limitation of this model. In the future, we would like to create a rodent model in which we can decrease blood flow by gradually thickening the VV walls, resulting in spontaneous aortic aneurysm.

The abdominal aorta is composed of the following three layers: the inner vascular wall (intima), medial layer (media), and outer vascular wall (adventitia), and of these, the adventitia has a unique blood supply system known as the vasa vasorum (VV). Aortic tissue is supplied with oxygen through adventitial VV perfusion and simple oxygen diffusion from the aortic blood flow1. However, geographically, the abdominal aorta has the least distribution of VVs compared to that in other parts of the aorta.2
A previous study reported on tissue hypoxia in human abdominal aortic aneurysm (AAA) walls with thick intraluminal thrombus (ILT)3. Additionally, it has been shown that an adventitial VV in aneurysmal walls is occluded with arteriosclerotic changes at a significantly higher rate, which is associated with tissue hypoxia in the AAA walls4. Based on these findings, a novel rodent model of AAA was created by inducing adventitial VV hypoperfusion5. In this model, VV hypoperfusion caused tissue hypoxia and the development of an infrarenal AAA, which had morphological and pathological characteristics similar to those of a human AAA6. Prime examples were the presence of ILTs and the accumulation of hyperplastic adipocytes6, and the potential to cause rupture7,8. These findings have been rarely observed in previous rodent models. Therefore, this model may greatly contribute to a deeper understanding of the mechanism responsible for AAA development and rupture. We introduce and describe a standard protocol used to induce AAAs through adventitial VV hypoperfusion, and we explain how to induce hypoxia in the aortic wall using surgical techniques.

<table><tbody><tr><td>rat</td><td>Japan SLC.Inc</td><td>Slc:SD rat</td><td>Sprague&amp;ndash;Dawley ratTM</td></tr><tr><td>povidone-iodine solution</td><td>Libatape Pharmaceutical Co., Ltd.</td><td>4987335 111457</td><td></td></tr><tr><td>5-0 silk string</td><td>Akiyama Medical MFG. CO.,LTD</td><td>JIS No.1</td><td></td></tr><tr><td>vascular clips</td><td>Natsume Seisakusho Co., Ltd.</td><td>C-42-S-2</td><td></td></tr><tr><td>polyurethane catheter (24-gauge indwelling needle)</td><td>MEDIKIT</td><td>24G</td><td>Supercath Z4VTM, 24-gauge indwelling needle</td></tr><tr><td>polyurethane catheter (18-gauge indwelling needle)</td><td>MEDIKIT</td><td>18G</td><td>Supercath Z3VTM, 18-gauge indwelling needle</td></tr><tr><td>8-0 monofilament string</td><td>Ethicon Suture</td><td>c-42-S-2</td><td>PROLENE Polypropylene Suture, Repair the incision with the suture</td></tr></tbody></table>

creation of a rodent model of abdominal aortic aneurysm by blocking adventitial vasa vasorum perfusion

The adventitial vasa vasorum (VV) provides oxygen and nourishment to the aortic wall. Hypoxia in the aortic wall can cause enlarged abdominal aortic aneurysms (AAAs). This article introduces and describes a standard protocol used to induce AAAs through adventitial VV hypoperfusion created with a combination of polyurethane catheter insertion into the aortic lumen and suture ligation of the infrarenal abdominal aorta.
The protocol involves the use of male rats weighing 300-400 g, which are provided food and water ad libitum. After laparotomy with a ventral midline abdominal incision, exfoliation of the aorta is performed, which blocks blood flow from the perivascular tissue. Aortotomy involving a small incision adjacent to the renal artery branches is performed, and a polyurethane catheter is inserted using an 18-gauge indwelling needle. After repairing the incision, tight ligation of the aorta over the catheter blocks VV blood flow from the proximal direction through the aortic wall without disturbing the aortic blood flow. This technique can induce an AAA with progressive aortic dilatation.
The greatest benefit of this model is that VV hypoperfusion causes tissue hypoxia and the development of an infrarenal AAA, which has morphological and pathological characteristics similar to those of a human AAA.

The described operative techniques create a novel animal model of a chronic aortic hypoxia-induced aneurysm by using a combination of polyurethane catheter insertion and suture ligation of the infrarenal abdominal aorta in rats. The rats described in the Protocol section were euthanized 28 days after the procedure. The aortas were harvested and imaged to visualize aneurysm formation. Figure 2 shows development of the fusiform AAA. The upper and lower ends of the aorta in ex vivo have a normal diameter without dilatation. Aortic diameters were measured using transabdominal ultrasonography (Figure 3). The diameter generally reaches its maximum size at about 14 days after the procedure; thereafter, it remains unchanged or slightly increases. Figure 4 shows the histopathological image of the aneurysm at its maximum diameter after EVG staining. The image of the tissue on day 28 (aneurysm) showed prominent degradation of elastic fibers compared to that on day 0.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55763/55763fig1.jpg" /> 
Figure 1: Surgical procedures to induce an abdominal aortic aneurysm (AAA). 
(A) The infrarenal aorta is exfoliated from the surrounding tissue. (B) A polyurethane catheter cut 10 mm long is inserted through a small incision in the aorta. (C) The incision is repaired with an 8-0 monofilament suture and blood flow is restored. (D) The aorta is ligated with a 5-0 silk suture over the inserted catheter. Scale bar = 5 mm. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55763/55763fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/55763/55763fig2.jpg" /> 
Figure 2: Postoperative representative results. 
Macroscopic view on postoperative day 28 showing development of fusiform abdominal aortic aneurysms. The elevated margin of the retroperitoneum corresponds to the outer edge of the aneurysm (broken lines; left). The upper and lower ends of the aorta in ex vivo are normal (right). Scale bar = 3 mm. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55763/55763fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/55763/55763fig3.jpg" /> 
Figure 3: Maximum aortic diameters measured using transabdominal ultrasonography. 
The aortic diameter steadily increased in this rat model. Aortic diameters are presented as a mean &#177; standard deviation (n = 12). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55763/55763fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/55763/55763fig4.jpg" /> 
Figure 4: Representative images of aneurysmal tissue with Elastica van Gieson staining. 
Histological evaluation with EVG staining showing the degenerative elastic lamina in the media and formation of an intraluminal thrombus 28 days after the procedure (right). Elastic fiber fragmentation in the aortic media and sparse collagen fiber in the aortic adventitia are observed on day 28. Day 0 is before the procedure (left). Scale bar = 500 &#181;m. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55763/55763fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Creation of a Rodent Model of Abdominal Aortic Aneurysm by Blocking Adventitial Vasa Vasorum Perfusion at JoVE.com

Creation of a Rodent Model of Abdominal Aortic Aneurysm by Blocking Adventitial Vasa Vasorum Perfusion

Polyurethane catheter insertion into the aortic lumen and suture ligation of the aorta induce chronic hypoxia due to hypoperfusion of the adventitial vasa vasorum. This article describes a novel animal model of abdominal aortic aneurysm (AAA) with characteristics similar to those of AAA in humans.

The adventitial vasa vasorum (VV) provides oxygen and nourishment to the aortic wall. Hypoxia in the aortic wall can cause enlarged abdominal aortic ...

creation-rodent-model-abdominal-aortic-aneurysm-blocking-adventitial

Nanyang Technological University

Research

JoVE Journal

Medicine

9.1K Views.  Hamamatsu University School of Medicine. Polyurethaan katheter inbrengen in de aorta lumen en hechtdraad Afbinding van de aorta induceren chronische hypoxie als gevolg van hypoperfusie van de adventitial Wasa vasorum. Dit artikel beschrijft een roman diermodel van abdominale aorta aneurysma (AAA) met eigenschappen die vergelijkbaar zijn met die van AAA bij de mens.

Video: Creation of a Rodent Model of Abdominal Aortic Aneurysm by Blocking Adventitial Vasa Vasorum Perfusion

Orthotopic Aortic Transplantation: A Rat Model to Study the Development of Chronic Vasculopathy

Research models of chronic rejection are essential to investigate pathobiological and pathophysiological processes during the development of transplant vasculopathy (TVP).
The commonly used animal model for cardiovascular chronic rejection studies is the heterotopic heart transplant model performed in laboratory rodents. This model is used widely in experiments since Ono and Lindsey (3) published their technique. To analyze the findings in the blood vessels, the heart has to be sectioned and all vessels have to be measured.
Another method to investigate chronic rejection in cardiovascular questionings is the aortic transplant model (1, 2). In the orthotopic aortic transplant model, the aorta can easily be histologically evaluated (2). The PVG-to-ACI model is especially useful for CAV studies, since acute vascular rejection is not a major confounding factor and Cyclosporin A (CsA) treatment does not prevent the development of CAV, similar to what we find in the clinical setting (4). A7-day period of CsA is required in this model to prevent acute rejection and to achieve long-term survival with the development of TVP.
This model can also be used to investigate acute cellular rejection and media necrosis in xenogeneic models (5).

This video demonstrates the orthotopic aortic transplant model as a simple model to study the development of transplant vasculopathy (TVP) in rats.

Research models of chronic rejection are essential to investigate pathobiological and pathophysiological processes during the development of transplant ...

Creation of Murine Experimental Abdominal Aortic Aneurysms with Elastase 

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

Creation of Murine Experimental Abdominal Aortic Aneurysms with Elastase

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

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

Biology

A New Murine Model of Endovascular Aortic Aneurysm Repair

Endovascular aneurysm exclusion is a validated technique to prevent aneurysm rupture. Long-term results highlight technique limitations and new aspects of Abdominal aortic aneurysm (AAA) pathophysiology. There is no abdominal aortic aneurysm endograft exclusion model cheap and reproducible, which would allow deep investigations of AAA before and after treatment. We hereby describe how to induce, and then to exclude with a covered coronary stentgraft an abdominal aortic aneurysm in a rat. The well known elastase induced AAA model was first reported in 19901 in a rat, then described in mice2. Elastin degradation leads to dilation of the aorta with inflammatory infiltration of the abdominal wall and intra luminal thrombus, matching with human AAA. Endovascular exclusion with small covered stentgraft is then performed, excluding any interactions between circulating blood and the aneurysm thrombus. Appropriate exclusion and stentgraft patency is confirmed before euthanasia by an angiography thought the left carotid artery. Partial control of elastase diffusion makes aneurysm shape different for each animal. It is difficult to create an aneurysm, which will allow an appropriate length of aorta below the aneurysm for an easy stentgraft introduction, and with adequate proximal and distal neck to prevent endoleaks. Lots of failure can result to stentgraft introduction which sometimes lead to aorta tear with pain and troubles to stitch it, and endothelial damage with post op aorta thrombosis. Giving aspirin to rats before stentgraft implantation decreases failure rate without major hemorrhage. Clamping time activates neutrophils, endothelium and platelets, and may interfere with biological analysis.

Histological and biochemical modifications after aneurysm endograft exclusion are still unclear. We describe a new model of endograft implantation on a murine aneurysm. Through thrombus analysis with and without persistant circulating blood stream interface, and new endovascular biomaterials evaluation, this model will help to better understand endovascular aneurysm exclusion pathobiology.

Endovascular aneurysm exclusion is a validated technique to prevent aneurysm rupture. Long-term results highlight technique limitations and new aspects of ...

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 ...

A Model of Cardiac Remodeling Through Constriction of the Abdominal Aorta in Rats

Heart failure is one of the leading causes of death worldwide. It is a complex clinical syndromethat includes fatigue, dyspnea, exercise intolerance, and fluid retention. Changes in myocardial structure, electrical conduction, and energy metabolism develop with heart failure, leading to contractile dysfunction, increased risk of arrhythmias, and sudden death. Hypertensive heart disease is one of the key contributing factors of cardiac remodeling associated with heart failure. The most commonly-used animal model mimicking hypertensive heart disease is created via surgical interventions, such as by narrowing the aorta. Abdominal aortic constriction is a useful experimental technique to induce a pressure overload, which leads to heart failure. The surgery can be easily performed, without the need for chest opening or mechanical ventilation. Abdominal aortic constriction-induced cardiac pathology progresses gradually, making this model relevant to clinical hypertensive heart failure. Cardiac injury and remodeling can be observed 10 weeks after the surgery. The method described here provides a simple and effective approach to produce a hypertensive heart disease animal model that is suitable for studying disease mechanisms and for testing novel therapeutics.

A rat model of abdominal aortic constriction that induces cardiac hypertrophy and remodeling is described. An efficient, highly-reproducible, and minimally-invasive method is used to provide a simple yet useful platform for research in myocardial hypertrophy and dysfunction.

Heart failure is one of the leading causes of death worldwide. It is a complex clinical syndromethat includes fatigue, dyspnea, exercise intolerance, and ...

Murine Cervical Aortic Transplantation Model using a Modified Non-Suture Cuff Technique

With the introduction of powerful immunosuppressive protocols, distinct advances are possible in the prevention and therapy of acute rejection episodes. However, only minor improvement in the long-term results of transplanted solid organs could be observed over the past decades. In this context, chronic allograft vasculopathy (CAV) still represents the leading cause of late organ failure in cardiac, renal and pulmonary transplantation.
Thus far, the underlying pathogenesis of CAV development remains unclear, explaining why effective treatment strategies are presently missing and emphasizing a need for relevant experimental models in order to study the underlying pathophysiology leading to CAV formation. The following protocol describes a murine heterotopic cervical aortic transplantation model using a modified non-suture cuff technique. In this technique, a segment of the thoracic aorta is interpositioned in the right common carotid artery. With the use of the non-suture cuff technique, an easy to learn and reproducible model can be established, minimizing the possible heterogeneity of sutured vascular micro anastomoses.

Here, we present a protocol of heterotopic aortic transplantation in mice using the non-suture cuff technique in a cervical murine model. This model can be used to study the underlying pathology of chronic allograft vasculopathy (CAV) and can help evaluate new therapeutic agents in order to prevent its formation.

With the introduction of powerful immunosuppressive protocols, distinct advances are possible in the prevention and therapy of acute rejection episodes. ...

Porcine Model of Infrarenal Abdominal Aortic Aneurysm

Large animal models to study abdominal aortic aneurysms are sparse. The purpose of this model is to create reproducible, clinically significant infrarenal abdominal aortic aneurysms (AAA) in swine. To achieve this, we use a combination of balloon angioplasty, elastase and collagenase, and a lysyl oxidase inhibitor, called &#946;-aminopropionitrile (BAPN), to create clinically significant infrarenal aortic aneurysms, analogous to human disease.
Noncastrated male swine are fed BAPN for 7 days prior to surgery to achieve a steady state in the blood. A midline laparotomy is performed and the infrarenal aorta is circumferentially dissected. An initial measurement is recorded prior to aneurysm induction with a combination of balloon angioplasty, elastase (500 units)/collagenase (8000 units) perfusion, and topical elastase application. Swine are fed BAPN daily until terminal procedure on either postoperative day 7, 14, or 28, at which time the aneurysm is measured, and tissue procured. BAPN + surgery pigs are compared to pigs that underwent surgery alone.
Swine treated with BAPN and surgery had a mean aortic dilation of 89.9% &#177; 47.4% at day 7, 105.4% &#177; 58.1% at day 14, and 113.5% &#177; 30.2% at day 28. Pigs treated with surgery alone had significantly smaller aneurysms compared to BAPN + surgery animals at day 28 (p &#60; 0.0003). The BAPN + surgery group had macroscopic and immunohistochemical evidence of end stage aneurysmal disease.
Clinically significant infrarenal AAA can be induced using balloon angioplasty, elastase/collagenase perfusion and topical application, supplemented with oral BAPN. This model creates large, clinically significant AAA with hallmarks of human disease. This has important implications for the elucidation of AAA pathogenesis and testing of novel therapies and devices for the treatment of AAA. Limitations of the model include variation in BAPN ingested by swine, quality of elastase perfusion, and cost of BAPN.&#160;

This novel model creates robust infrarenal abdominal aortic aneurysms in swine using a combination of balloon angioplasty, elastase/collagenase perfusion, topical elastase application, and oral compound &#946;-aminopropionitrile administration, which interferes with collagen cross-linking.

Large animal models to study abdominal aortic aneurysms are sparse. The purpose of this model is to create reproducible, clinically significant infrarenal ...

Investigating the Molecular Mechanisms Underlying Murine Abdominal Aortic Aneurysm

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

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

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

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

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

Mouse Abdominal Aortic Aneurysm Model Induced by Perivascular Application of Elastase

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

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

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

Murine Model of Thoracic Aortic Dissection Induced by Oral &#946;-Aminopropionitrile and Subcutaneous Angiotensin II Infusion

Thoracic aortic dissection (TAD) is a highly fatal cardiovascular disease that lacks efficient medical treatment. Replication of animal models of TAD pathophysiology is essential for studying the intrinsic mechanisms of TAD. The widely used TAD model induced by &#946;-aminopropionitrile (BAPN, an irreversible and orally active lysyl oxidase inhibitor) in mice has the limitation of an inconsistent success rate. This protocol describes in detail a reported modified murine model of TAD induced by oral BAPN combined with subcutaneous angiotensin II (Ang II) infusion. After four weeks of BAPN administration followed by 24 h of Ang II infusion, a murine model with characteristics similar to human TAD was reliably induced, and the success rate of TAD model construction was significantly improved. Oral BAPN inhibits the cross-linking of elastin and collagen, resulting in the destruction of the aortic wall structure and inducing aortic dilation and dissection formation to a certain extent. The subsequent induction of Ang II further exacerbates the degeneration of the aortic wall, thereby promoting the occurrence of TAD. Consequently, the combination of BAPN and Ang II represents a refined approach to constructing a murine TAD model, offering a valuable tool to explore the pathogenesis and potential therapeutic approaches for TAD.

This protocol provides a detailed, step-by-step procedure for the induction of thoracic aortic dissection in mice. Specifically, it includes the precise calculation of the required doses of &#946;-aminopropionitrile and angiotensin II, the procedure for osmotic pump filling, and the implantation technique of the osmotic pump.

Thoracic aortic dissection (TAD) is a highly fatal cardiovascular disease that lacks efficient medical treatment. Replication of animal models of TAD ...

Creation of a Rodent Model of Abdominal Aortic Aneurysm by Blocking Adventitial Vasa Vasorum Perfusion

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