This work was supported by the National Natural Science Foundations of China (No. 82300542, 81770471), Sichuan Science and Technology Program (No. 2022YFS0359, 2019JDRC0104) and Post-Doctor Research Project, West China Hospital, Sichuan University (No. 2023HXBH108). The funding bodies played no role in the design of the study, the collection, analysis, and interpretation of the data, and the writing of the manuscript.

The animal experiment protocol complied with the National Institute of Health Guide for the Care and Use of Laboratory Animals (NIH Pub No. 86/23, 1985) and was approved by the Institutional Animal Care and Use Committee of West China Hospital, Sichuan University (Approval Number 202311300012). Eight to ten-week-old C57/B6J male mice were used for this study. The details of the reagents and equipment used are listed in the Table of Materials.
1. Preoperative preparation
<ol>
	<li>Obtain the animals and feed them a normal chow diet with clean water under standard conditions (temperature 24 &#176;C, humidity 40%-50%, 12-h light-dark cycle).</li>
	<li>Cut a cotton pad into 40 mm x 4 mm strips and sterilize it along with scissors, forceps, and other surgical equipment.</li>
	<li>Place the animal into the induction chamber of the anesthesia machine and use 3%-3.5% isoflurane to quickly induce anesthesia (following institutionally approved protocols). Additionally, prescribe butorphanol (1 mg/kg, intramuscular injection) as pre-emptive analgesia medication.
	<ol>
		<li>Remove the mouse from the chamber when it no longer responds to pain stimulation, then place a mask on the mouse and maintain the depth of anesthesia with 1%-1.5% isoflurane. Monitor respiratory rate and depth every 5-10 min.</li>
	</ol>
	</li>
	<li>Place the mouse in a supine position on the heating pad, use adhesive tape to immobilize the limbs, and apply eye ointment to prevent dryness.</li>
	<li>Use an electric shaver or hair removal lotion to completely remove the hair in the abdominal area, then disinfect this area with povidone-iodine and ethanol-soaked cotton swabs in a circular motion at least three times.</li>
</ol>
2. Surgical procedure
<ol>
	<li>Place the mouse under a stereomicroscope. Use scissors to make a longitudinal incision of approximately 2-2.5 cm along the midline of the abdomen.</li>
	<li>Enter the peritoneal cavity through the linea alba (transabdominal approach), place a retractor to expose the peritoneal cavity, and pack the intestines and colon into the right half of the abdomen using normal saline-soaked gauze.</li>
	<li>Perform blunt separation of connective and adipose tissue adjacent to the infrarenal abdominal aorta and inferior vena cava (IVC) using forceps and cotton swabs. Gently dissect the abdominal aorta and expose the gap between the abdominal aorta and the overlying psoas major, as previously referenced25.
	<ol>
		<li>Insert the sterilized cotton pad strips into that gap, being extremely careful not to damage the lumbar artery or vein. It is not necessary to isolate the abdominal aorta from the IVC.</li>
	</ol>
	</li>
	<li>Soak the cotton ball with 0.9 M CaCl2 and place it on the adventitia of the abdominal aorta for 15 min. Use sterilized gauze to cover the abdominal cavity during the process of infiltration.</li>
	<li>Gently remove the CaCl2-soaked cotton ball and wash the infiltrated segment with 0.9% saline solution.</li>
	<li>Dilute the elastase to 2 mg/mL (8 U/mL) with ddH2O. Use an insulin syringe to apply 50 &#181;L of diluted elastase to the infrarenal segment of the abdominal aorta and wrap the cotton pad strips to cover the abdominal aorta and IVC. Use sterilized gauze to cover the abdominal cavity during the process of infiltration. In the Sham group, use 0.9% normal saline to replace CaCl2 and PPE.</li>
	<li>15 min later, carefully remove the gauze and cotton pad strips. Move the intestines and colon back to their original positions, cover the treated aorta with the mesentery, and moisten the intestines to prevent adhesion.</li>
	<li>Close the muscle layer and skin separately using 6-0 nonabsorbable polypropylene suture. Disinfect the surgical area with povidone-iodine and ethanol.</li>
	<li>Place the mouse on a heating pad and monitor its respiratory status until it regains consciousness. Record the operation time and the number of experimental animals. Prescribe carprofen (5 mg/kg, subcutaneously) as post-operative analgesia for at least 3 days after the surgery, according to veterinary recommendation.</li>
</ol>
3. Ultrasound examination
<ol>
	<li>21 days after the surgery, conduct an ultrasound examination to evaluate the incidence rate of AAA.</li>
	<li>Anesthetize the mouse (following institutionally approved protocols) and remove the abdominal hair as previously described.</li>
	<li>Place the mouse on the heating pad of the rodent ultrasound machine. Apply ultrasonic couplant to the abdomen and measure the maximum diameter of the aneurysm and the diameter of the normal segment of the abdominal aorta.
	<ol>
		<li>Calculate the increased percentage of the infrarenal abdominal aorta. The criterion for successful AAA formation is that the maximum diameter of the AAA has increased by 50% or more compared to the diameter of the normal segment of the infrarenal abdominal aorta.</li>
	</ol>
	</li>
	<li>Use a napkin to wipe off the remaining ultrasonic couplant after the examination, and place the mouse on the heating pad until it has fully recovered.</li>
</ol>
4. Abdominal aorta harvest and pathological experiments
<ol>
	<li>Sacrifice the mouse by isoflurane overdose on the day following the ultrasound examination (following institutionally approved protocols).</li>
	<li>Open the thoracic and abdominal cavities and perform perfusion with 1x PBS through the left ventricle until the lungs and liver turn white, following the procedure described in previous literature27.</li>
	<li>Use a digital caliper to measure the external diameter of the infrarenal aorta. Harvest the infrarenal abdominal aorta under a stereomicroscope and store the vessel in 4% paraformaldehyde for 24-48 h.</li>
	<li>Remove the connective and adipose tissue surrounding the abdominal aorta. Embed the aorta in paraffin to prepare sections, cutting the sections into 3-5 &#181;m slices as previously described18,28.</li>
	<li>Perform hematoxylin and eosin (H&#38;E), Masson&#39;s trichrome, and Elastic-Van Gieson (EVG) staining on the deparaffinized slices using respective staining kits according to the manufacturer&#39;s instructions.
	<ol>
		<li>Analyze the vessel structure and inflammation with H&#38;E staining, observe extracellular matrix (ECM) degradation and collagen fibers with Masson&#39;s trichrome staining, and examine elastic fibers with EVG staining.</li>
	</ol>
	</li>
</ol>

Investigating the Molecular Mechanisms Underlying Murine Abdominal Aortic Aneurysm

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The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Research into the molecular mechanisms of AAA requires a stable animal model. Consequently, numerous AAA animal models have been established since its initial development by Economou et al. in the 1960s29. Among these models, CaCl2 is frequently employed in rodents due to its cost-effectiveness, technical simplicity, and reliable reproducibility. However, perivascular CaCl2 infiltration has been shown to be unstable in establishing AAA. Bi et al. and Freestone et al. reported...

Abdominal aortic aneurysm (AAA) is defined as a diameter increase of more than 50% or a maximum aortic diameter exceeding 3 cm in the abdominal aorta. This condition poses a significant threat to life, with around a 90% mortality rate upon aneurysm rupture1,2,3. Currently, open surgical repair and endovascular aortic repair (EVAR) are the only available interventions for AAA patients4,5,6. However, there is insufficient evidence to support the effectiveness of medical treatments in inhibiting aneurysm formation or slowing the growth rate of the abdominal aorta in patients who do not have surgical indications7. Nevertheless, non-specific treatments, including persistent surveillance of maximum aneurysm diameter, blood pressure control, antiplatelet therapy, and statins, are used to reduce the risk of sudden aneurysm rupture and potential cardiovascular and neurological events as much as possible. Despite this, the role of antiplatelet medications and statins in preventing aneurysm rupture remains controversial and requires further studies5,7,8,9,10.
A stable AAA animal model is vital for investigating the pathogenesis of AAA, and numerous methods have been introduced to establish such a model11,12,13. Currently, calcium chloride (CaCl2), elastase, angiotensin II (Ang II), xenografts, and transgenic models are used in establishing rodent AAA models11,12,13. Among these, Ang II is the most commonly used in mice, while CaCl2 and elastase are also widely used in mice and rats11,12,13,14. The Ang II-induced AAA model is the sole animal model capable of inducing atherosclerosis that closely resembles human AAA pathology, which is simple and reproducible and obviates the need for laparotomy11,12,13. However, in contrast to the models induced by CaCl2 or elastase, the site of aneurysms induced by Ang II is uncertain and frequently observed in the descending aorta or suprarenal abdominal aorta, with a heightened risk of rupture11,12,13,14. The aneurysm incidence rate of the Ang II-induced AAA model in gene-deficient mice can be as high as 100%, while only 39% of C57BL/6 mice developed aneurysms, and the time and economic cost of obtaining gene-deficient mice are high13,15.
Initially introduced by Gertz et al., CaCl2 was utilized to induce aneurysm formation in the carotid artery and later modified by Chiou et al. to establish an AAA model in mice16,17. However, despite its ability to induce elastic fiber breakdown, vessel inflammation, and extracellular matrix degradation, CaCl2 lacks several human AAA features, including atherothrombosis, intraluminal thrombus (ILT), and aneurysm rupture12,18. In addition, the formation rate and diameter increase percentage of periaortic CaCl2 application remain unstable13,19. The initial intraluminal porcine pancreatic elastase (PPE) perfusion model for inducing AAA was developed by Anidjar et al. in 1990, followed by Bhamidipati et al.&#39;s report on periadventitial PPE infiltration in a murine model20,21. Nowadays, the majority of elastase-induced AAA animal models utilize Bhamidipati&#39;s protocol due to its feasibility and minimally invasive nature. However, it is important to note that the incidence rate and maximum diameter of AAA can vary and are generally lower than intra-aortic PPE perfusion. Moreover, aneurysms induced by periaortic application of elastase present a tendency to heal spontaneously11,12,20,21,22,23.
To address these limitations, Tanaka et al. proposed a combination approach involving intraluminal PPE perfusion and CaCl2 infiltration to establish an AAA model in rats, which yielded satisfactory results24. In addition, Zhu et al. demonstrated that the PPE + CaCl2 model offers advantages such as higher survival rates and increased aneurysm formation rates compared to both the single PPE group and the PPE + BAPN group25. This combined approach also exhibits good stability and reproducibility. Moreover, Bi et al. successfully established a rabbit AAA model via the combination of periaortic CaCl2 and elastase incubation. The average dilation ratio was 65.3% &#177; 8.9% on day 5, which further increased to 86.5% &#177; 28.7% on day 15 and significantly escalated to 203.6% &#177; 39.1% on day 3026. However, there is currently no existing literature reporting the induction of AAA in rodents by combining periaortic CaCl2 and elastase application.
This manuscript presents a standard protocol for establishing a murine AAA model through the combined use of periadventitial CaCl2 and elastase infiltration. The subsequent sections provide detailed surgical procedures and present representative results of the murine AAA model.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Anesthesia Machine</td>
			<td>RWD</td>
			<td>R550</td>
			<td></td>
		</tr>
		<tr>
			<td>Butorphanol</td>
			<td>Jiangsu Hengrui Pharmaceutical Co., Ltd</td>
			<td>220608BP</td>
			<td>1ml: 1mg</td>
		</tr>
		<tr>
			<td>C57BL/6JGpt Male Mice</td>
			<td>GemPharmatech</td>
			<td>N000013</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium Chloride</td>
			<td>Sigma Aldrich</td>
			<td>C4901</td>
			<td>100 g</td>
		</tr>
		<tr>
			<td>Carprofen</td>
			<td>MCE</td>
			<td>HY-B1227</td>
			<td>100 mg</td>
		</tr>
		<tr>
			<td>Chow Diet</td>
			<td>Dossy Experimental Animals Co.Ltd</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Digital Caliper</td>
			<td>Greener</td>
			<td>IP54</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Jinhe Pharmaceutical Co.Ltd</td>
			<td>539682</td>
			<td>75%/500 mL</td>
		</tr>
		<tr>
			<td>EVG Staining Kit</td>
			<td>Solarbio</td>
			<td>G1597</td>
			<td></td>
		</tr>
		<tr>
			<td>GraphPad Prism</td>
			<td>Graphpad</td>
			<td></td>
			<td>Ver 9.0.0</td>
		</tr>
		<tr>
			<td>H&#38;E Staining Kit</td>
			<td>Servicebio</td>
			<td>G1076</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin Syringe</td>
			<td>BD</td>
			<td>2143420</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>RWD</td>
			<td>R510-22-4</td>
			<td>100 mL</td>
		</tr>
		<tr>
			<td>Masson Staining Kit</td>
			<td>Servicebio</td>
			<td>G1006</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal Saline</td>
			<td>Servicebio</td>
			<td>G4702</td>
			<td>500 mL</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Biosharp</td>
			<td>BL539A</td>
			<td>4%/500 mL</td>
		</tr>
		<tr>
			<td>PBS, 1x</td>
			<td>Servicebio</td>
			<td>G4202</td>
			<td>500 mL</td>
		</tr>
		<tr>
			<td>Porcine Pancreatic Elastase</td>
			<td>Sigma Aldrich</td>
			<td>E1250</td>
			<td>100 mg</td>
		</tr>
		<tr>
			<td>Povidine Iodine</td>
			<td>Yongan Pharmaceutical Co.Ltd</td>
			<td></td>
			<td>5%/100 mg</td>
		</tr>
		<tr>
			<td>Prolene Polypropylene Suture</td>
			<td>Ethicon LLC</td>
			<td>8709H</td>
			<td></td>
		</tr>
		<tr>
			<td>Rodent Ultrasound System</td>
			<td>Fujifilm</td>
			<td>Vevo 3100LT</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereo Microscope</td>
			<td>Olympus</td>
			<td>SZ61</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrasonic Couplant</td>
			<td>Keppler</td>
			<td>KL-250</td>
			<td>250 g</td>
		</tr>
	</tbody>
</table>

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.

In this study, a total of 24 mice were included and randomly assigned: 12 in the Sham group and 12 in the PPE + CaCl2 group, respectively. All data are presented as means &#177; standard deviations unless otherwise stated. The average operation time was 55.67 min &#177; 4.08 min. There were no intra-operative deaths or aneurysm ruptures, and the survival rate within 21 days after surgery was 100%. No severe intestinal adhesions or complications related to abdominal aorta dissection were observed.
<p class=...

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.

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 ...

a-mouse-abdominal-aortic-aneurysm-model-periadventitial-calcium

Research

JoVE Journal

Medicine

642 Views.  Sichuan University. Deze studie presenteert een protocol voor het opstellen van een abdominaal aorta-aneurysmamodel bij muizen met behulp van calciumchloride en pancreaselastase van varkens, dat kan worden gebruikt om het pathofysiologische mechanisme onder abdominaal aorta-aneurysma te onderzoeken. Ons protocol combineert de voordelen van periadventitiële PBM en de calciumchloride-infiltratie, een veilig, stabiel en reproduceerbaar AAA-model voor muizen, dat een hoge incidenti...

Video: Auteur in de schijnwerpers: ontwikkeling en karakterisering van een muismodel voor abdominaal aorta-aneurysma

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 ...

Ferric Chloride-induced Thrombosis Mouse Model on Carotid Artery and Mesentery Vessel

Severe thrombosis and its ischemic consequences such as myocardial infarction, pulmonary embolism and stroke are major worldwide health issues. The ferric chloride injury is now a well-established technique to rapidly and accurately induce the formation of thrombi in exposed veins or artery of small and large diameter. This model has played a key role in the study of the pathophysiology of thrombosis, in the discovery and validation of novel antithrombotic drugs and in the understanding of the mechanism of action of these new agents. Here, the implementation of this technique on a mesenteric vessel and carotid artery in mice is presented. The method describes how to label circulating leukocytes and platelets with a fluorescent dye and to observe, by intravital microscopy on the exposed mesentery, their accumulation at the injured vessel wall which leads to the formation of a thrombus. On the carotid artery, the occlusion caused by the clot formation is measured by monitoring the blood flow with a Doppler probe.

The FeCl3 induced thrombosis model in mice is described herein. A method to monitor thrombus growth by intravital microscopy observation on a mesenteric vessel and by blood flow measurement in the carotid artery is presented.

Severe thrombosis and its ischemic consequences such as myocardial infarction, pulmonary embolism and stroke are major worldwide health issues. The ferric ...

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 ...

Using the Sleeve Technique in a Mouse Model of Aortic Transplantation - An Instructional Video

Orthotopic aortic transplantation using the sleeve technique reduces injury to the aorta with failure rate of only 10-20%. The time to anastomose the aorta in mice using the sleeve method was short and easy averaging 20 min, permitting studies of iso/allo grafts. The following article describes the aortic transplantation procedure used in our laboratory. The mice were anesthetized with a mixture of 1.5% volume isoflurane and 100% oxygen through a face mask. At this point, the segment of the aorta between the renal arteries and its bifurcation was separated from the vena cava, freely prepared and clampedat the proximal and distal segments with a single silk suture. Prior to the removal of the aorta, a saline solution containing heparin was injected into the inferior vena cava. Then the aorta was cut between the clamps and a saline heparin solution was used to flush the lumen. The sleeve technique with monofilament sutures was used in order to transplant the abdominal aorta in the orthotopic position.

We present an orthotopic aortic transplantation model using the sleeve technique in mice. It is a very rapid anastomosis method, which can be employed in studies of vascular disease.

Orthotopic aortic transplantation using the sleeve technique reduces injury to the aorta with failure rate of only 10-20%. The time to anastomose the ...

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.

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 ...

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 ...

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 ...

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 ...

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

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