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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+abdominal+and+thoracic+aortic+medial+structure+in+mammals.+Deviation+of+man+from+the+usual+pattern.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lymphangiogenesis+and+angiogenesis+in+abdominal+aortic+aneurysm.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Association+of+intraluminal+thrombus+in+abdominal+aortic+aneurysm+with+local+hypoxia+and+wall+weakening.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adventitial+vasa+vasorum+arteriosclerosis+in+abdominal+aortic+aneurysm.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoperfusion+of+the+Adventitial+Vasa+Vasorum+Develops+an+Abdominal+Aortic+Aneurysm.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+Mass+Spectrometry+Reveals+a+Unique+Distribution+of+Triglycerides+in+the+Abdominal+Aortic+Aneurysmal+Wall.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+preventive+effect+of+fish+oil+on+abdominal+aortic+aneurysm+development.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adipocyte+in+vascular+wall+can+induce+the+rupture+of+abdominal+aortic+aneurysm.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biological+scaffolds+for+heart+valve+tissue+engineering.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+vasa+vasorum+in+nourishment+of+the+aortic+wall.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regression+of+abdominal+aortic+aneurysm+by+inhibition+of+c-Jun+N-terminal+kinase.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Creation+of+murine+experimental+abdominal+aortic+aneurysms+with+elastase.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subcutaneous+Angiotensin+II+Infusion+using+Osmotic+Pumps+Induces+Aortic+Aneurysms+in+Mice.">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.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endovascular+versus+open+repair+of+abdominal+aortic+aneurysm.">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 border="0" cellpadding="0" cellspacing="0" width="826">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:292px;">rat</td>
			<td style="width:115px;">Japan SLC.Inc</td>
			<td style="width:185px;">Slc:SD rat</td>
			<td style="width:235px;">Sprague&#8211;Dawley ratTM</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:292px;">povidone-iodine solution</td>
			<td style="width:115px;">Libatape Pharmaceutical Co., Ltd.</td>
			<td style="width:185px;">4987335 111457</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:292px;">5-0 silk string</td>
			<td style="width:115px;">Akiyama Medical MFG. CO.,LTD</td>
			<td style="width:185px;">JIS No.1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">vascular clips</td>
			<td>Natsume Seisakusho Co., Ltd.</td>
			<td>C-42-S-2</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:292px;">polyurethane catheter (24-gauge indwelling needle)</td>
			<td style="width:115px;">MEDIKIT</td>
			<td style="width:185px;">24G</td>
			<td>Supercath Z4VTM, 24-gauge indwelling needle</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:292px;">polyurethane catheter (18-gauge indwelling needle)</td>
			<td style="width:115px;">MEDIKIT</td>
			<td style="width:185px;">18G</td>
			<td>Supercath Z3VTM, 18-gauge indwelling needle</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:292px;">8-0 monofilament string</td>
			<td style="width:115px;">Ethicon Suture</td>
			<td style="width:185px;">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/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/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/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/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 ...

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9.0K Views.  Hamamatsu University School of Medicine. 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.

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

Creation of Reversible Cholestatic Rat Model

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

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

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

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 ...

Reproducable Paraplegia by Thoracic Aortic Occlusion in a Murine Model of Spinal Cord Ischemia-reperfusion

Background 
Lower extremity paralysis continues to complicate aortic interventions. The lack of understanding of the underlying pathology has hindered advancements to decrease the occurrence this injury.&#160;The current model demonstrates reproducible lower&#160;extremity paralysis following thoracic aortic occlusion.
Methods 
Adult male C57BL6 mice were anesthetized with isoflurane. Through a cervicosternal incision the aorta was exposed. The descending thoracic aorta and left subclavian arteries were identified without entrance into pleural space. Skeletonization of these arteries was followed by immediate closure (Sham) or occlusion for 4 min (moderate ischemia) or 8 min (prolonged ischemia). The sternotomy and skin were closed and the mouse was transferred to warming bed for recovery.&#160; Following recovery, functional analysis was obtained at 12 hr intervals until 48 hr.
Results 
Mice that underwent sham surgery showed no observable hind&#160;limb deficit. Mice subjected to moderate ischemia for 4 min had minimal functional deficit at 12 hr followed by progression to complete paralysis at 48 hr. Mice subjected to prolonged ischemia had an immediate paralysis with no observable hind-limb movement at any point in the postoperative period. There was no observed intraoperative or post&#160;operative mortality.
Conclusion 
Reproducible lower extremity paralysis whether immediate or delayed can be achieved in a murine model. Additionally, by using a median sternotomy and careful dissection, high survival rates, and reproducibility can be achieved.

The lack of mechanistic understanding of spinal cord ischemia-reperfusion injury has hindered further adjuncts to prevent paraplegia following high risk aortic operations. Thus, the development of animal models is imperative. This manuscript demonstrates reproducible lower extremity paralysis following thoracic aortic occlusion in a murine model.

Background
Lower extremity paralysis continues to complicate aortic interventions. The lack of understanding of the underlying pathology has hindered ...

Ascending Aortic Constriction in Rats for Creation of Pressure Overload Cardiac Hypertrophy Model

Ascending aortic constriction is the most common and successful surgical model for creating pressure overload induced cardiac hypertrophy and heart failure. Here, we describe a detailed surgical procedure for creating pressure overload and cardiac hypertrophy in rats by constriction of the ascending aorta using a small metallic clip. After anesthesia, the trachea is intubated by inserting a cannula through a half way incision made between two cartilage rings of trachea. Then a skin incision is made at the level of the second intercostal space on the left chest wall and muscle layers are cleared to locate the ascending portion of aorta. The ascending aorta is constricted to 50&#8211;60% of its original diameter by application of a small sized titanium clip. Following aortic constriction, the second and third ribs are approximated with prolene sutures. The tracheal cannula is removed once spontaneous breathing was re-established. The animal is allowed to recover on the heating pad by gradually lowering anesthesia. The intensity of pressure overload created by constriction of the ascending aorta is determined by recording the pressure gradient using trans-thoracic two dimensional Doppler-echocardiography. Overall this protocol is useful to study the remodeling events and contractile properties of the heart during the gradual onset and progression from compensated cardiac hypertrophy to heart failure stage.

We describe a stepwise procedure for creating pressure overload and left ventricular hypertrophy in Wistar rats by constriction of the ascending aorta using a small metallic clip. This model is extensively used for studying remodeling changes during cardiac hypertrophy and for identifying and evaluating strategies for regression of such changes.

Ascending aortic constriction is the most common and successful surgical model for creating pressure overload induced cardiac hypertrophy and heart ...

Creation of Abdominal Adhesions in Mice

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

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

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

Reproducible Arterial Denudation Injury by Infrarenal Abdominal Aortic Clamping in a Murine Model

Percutaneous vascular interventions uniformly result in arterial denudation injuries that subsequently lead to thrombosis and restenosis. These complications can be attributed to impairments in re-endothelialization within the wound margins. Yet, the cellular and molecular mechanisms of re-endothelialization remain to be defined. While several animal models to study re-endothelialization after arterial denudation are available, few are performed in the mouse because of surgical limitations. This undermines the opportunity to exploit transgenic mouse lines and investigate the contribution of specific genes to the process of re-endothelialization. Here, we present a step-by-step protocol for creating a highly reproducible murine model of arterial denudation injury in the infrarenal abdominal aorta using external vascular clamping. Immunocytochemical staining of injured aortas for fibrinogen and &#946;-catenin demonstrate the exposure of a pro-thrombotic surface and the border of intact endothelium, respectively. The method presented here has the advantages of speed, excellent overall survival rate, and relative technical ease, creating a uniquely practical tool for imposing arterial denudation injury in transgenic mouse models. Using this method, investigators may elucidate the mechanisms of re-endothelialization under normal or pathological conditions.

Understanding the cellular and molecular mechanisms of re-endothelialization following arterial denudation injury is of paramount importance in preventing thrombosis and restenosis of arteries. Here we describe a protocol for reproducible arterial denudation injury of the infrarenal abdominal aorta. The procedure was developed to investigate the underlying mechanisms that regulate endothelial regeneration using mouse models.

Percutaneous vascular interventions uniformly result in arterial denudation injuries that subsequently lead to thrombosis and restenosis. These ...

Quantitative Micro-CT Analysis of Aortopathy in a Mouse Model of &#946;-aminopropionitrile-induced Aortic Aneurysm and Dissection

Aortic aneurysm and dissection is associated with significant morbidity and mortality in the population and can be highly lethal. While animal models of aortic disease exist, in vivo imaging of the vasculature has been limited. In recent years, micro-computerized tomography (micro-CT) has emerged as a preferred modality for imaging both large and small vessels both in vivo and ex vivo. In conjunction with a method of vascular casting, we have successfully used micro-CT to characterize the frequency and distribution of aortic pathology in &#946;-aminopropionitrile-treated C57/Bl6 mice. Technical limitations of this method include variations in the quality of the perfusion introduced by poor animal preparation, the application of proper methodologies for vessel size quantification, and the non-survivability of this procedure. This article details a methodology for the intravascular perfusion of a lead-based radiopaque silicone rubber for the quantitative characterization of aortopathy in a mouse model of aneurysm and dissection. In addition to visualizing aortic pathology, this method may be used for examining other vascular beds in vivo or vascular beds removed post-mortem.

This article describes a detailed methodology of using a radiopaque lead-based silicone rubber to perfuse the murine vasculature for aortic diameter quantification in a mouse model of aortic aneurysm and dissection.

Aortic aneurysm and dissection is associated with significant morbidity and mortality in the population and can be highly lethal. While animal models of ...

Ultrasound Imaging of the Thoracic and Abdominal Aorta in Mice to Determine Aneurysm Dimensions

Contemporary high-resolution ultrasound instruments have sufficient resolution to facilitate the measurement of mouse aortas. These instruments have been widely used to measure aortic dimensions in mouse models of aortic aneurysms. Aortic aneurysms are defined as permanent dilations of the aorta, which occur most frequently in the ascending and abdominal regions. Sequential measurements of aortic dimensions by ultrasound are the principal approach for assessing the development and progression of aortic aneurysms in vivo. Although many reported studies used ultrasound imaging to measure aortic diameters as a primary endpoint, there are confounding factors, such as probe position and cardiac cycle, that may impact the accuracy of data acquisition, analysis, and interpretation. The purpose of this protocol is to provide a practical guide on the use of ultrasound to measure the aortic diameter in a reliable and reproducible manner. This protocol introduces the preparation of mice and instruments, the acquisition of appropriate ultrasound images, and data analysis.

Ultrasound imaging has become a common modality to determine the luminal dimensions of thoracic and abdominal aortic aneurysms in mice. This protocol describes the procedure to acquire reliable and reproducible two-dimensional ultrasound images of the ascending and abdominal aorta in mice.

Contemporary high-resolution ultrasound instruments have sufficient resolution to facilitate the measurement of mouse aortas. These instruments have been ...

Murine Surgical Model of Topical Elastase Induced Descending Thoracic Aortic Aneurysm

According to the Center for Disease Control, aortic aneurysms (AAs) were considered a leading cause of death in all races and both sexes from 1999-2016. An aneurysm forms as a result of progressive weakening and eventual dilation of the aorta, which can rupture or tear once it reaches a critical diameter. Aneurysms of the descending aorta in the chest, called descending thoracic aortic aneurysms (dTAA), make up a large proportion of aneurysm cases in the United States. Uncontained dTAA rupture is almost universally lethal, and elective repair has a high rate of morbidity and mortality. The purpose of our model is to study dTAA specifically, to elucidate the pathophysiology of dTAA and to search for molecular targets to halt the growth or reduce the size of dTAA. By having a murine model to study thoracic pathology precisely, targeted therapies can be developed to specifically test dTAA. The method is based on the placement of porcine pancreatic elastase (PPE) directly on the outer murine aortic wall after surgical exposure. This creates a destructive and inflammatory reaction, which weakens the aortic wall and allows for aneurysm formation over weeks to months. Though murine models possess limitations, our dTAA model produces robust aneurysms of predictable size. Furthermore, this model can be used to test genetic and pharmaceutical targets which may arrest dTAA growth or prevent rupture. In human patients, interventions such as these could help avoid aneurysm rupture, and difficult surgical intervention.

We describe a surgical protocol to consistently induce robust descending thoracic aortic aneurysms in mice. The procedure involves left thoracotomy, thoracic aorta exposure, and placement of a sponge soaked in porcine pancreatic elastase on the aortic wall.

According to the Center for Disease Control, aortic aneurysms (AAs) were considered a leading cause of death in all races and both sexes from 1999-2016. ...

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 ...