This research was supported by funding from the National Natural Science Foundation of China (NSFC, 81730010, 91839302, 81921001, 31930056, and 91529203) and the National Key R&#38;D Program of China (2019YFA 0801600).

Animal studies were performed in compliance with the guidelines of the Institutional Animal Care and Use Committee of Peking University Health Science Center and were approved by the Biomedical Ethics Committee of Peking University (LA2015142). All the mice for surgery were anesthetized with isoflurane (1.5%-2%), and anesthesia was carefully monitored to avoid pain or discomfort for the mice.
1. Preparation
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	<li>Cut 0.3 cm wide strips of powder-free rubber gloves and ga...

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J., Kipke, D. R., Shain, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole+animal+perfusion+fixation+for+rodents.">Whole animal perfusion fixation for rodents.</a> Journal of Visualized Experiments. (65), e3564 (2012).</li><li>Yu, B., 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=CYLD+deubiquitinates+nicotinamide+adenine+dinucleotide+phosphate+oxidase+4+contributing+to+adventitial+remodeling.">CYLD deubiquitinates nicotinamide adenine dinucleotide phosphate oxidase 4 contributing to adventitial remodeling.</a> Arteriosclerosis, Thrombosis, and Vascular Biology. 37 (8), 1698-1709 (2017).</li><li>Altobelli, E., Rapacchietta, L., Profeta, V. 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Periaortic application of CaPO4 is a robust approach to induce AAA in mice. Several studies have used the CaPO4 model and consistently reported that this is a rapid and reproducible method to study AAA in mice7,9. This model is considered to recapitulate part of the features of human aortic aneurysm and provide mechanistic insights into AAA pathogenesis, including inflammation and extracellular matrix degradation.
<p class="jove_content"...

An abdominal aortic aneurysm (AAA) is a lethal cardiovascular disease characterized by permanent dilation of the abdominal aorta, with high mortality rates once rupture occurs. AAA is associated with aging, smoking, male gender, hypertension, and hyperlipidemia1. Several pathological processes have been shown to contribute to AAA formation, including extracellular matrix fiber proteolysis, immune cell infiltration, and loss of vascular smooth muscle cells. Currently, the pathological mechanisms of AAA remain elusive, and there are no proven drugs for the treatment of AAA1. Research into human AAA is limited due to the existence of few human aortic samples; thus, several chemical modification-induced animal AAA models have been established and widely adopted, including subcutaneous angiotensin II (AngII) infusion, perivascular or intraluminal elastase incubation, and perivascular calcium phosphate application2. A commonly used mouse model is the application of calcium phosphate (CaPO4) to the adventitia of the infrarenal abdominal aorta, which is cost-effective and does not require genetic modification.
Direct periaortic application of CaCl2 to the carotid artery of rabbits to induce aneurysmal change was initially reported by Gertz et al.3 and was later applied to the abdominal aortas of mice. The model was developed by Yamanouchi et al. to accelerate aortic dilation by using CaPO4 crystals in mice4. Infiltration of CaPO4 into mice aortas recapitulates many pathological features observed in human AAAs, including profound macrophage infiltration, extracellular matrix degradation, and calcium deposition. The risk factors of human AAA, such as hyperlipidemia, also augment CaPO4-induced AAA in mice5. In contrast to AngII perfusion-induced AAA in ApoE-/- or LDLR-/- mice, CaPO4-induced AAA occurs in the infrarenal aortic region, which mimics human AAA. Currently, this method has been widely applied to assess susceptibility to AAA development in genetically modified mice and evaluate the anti-AAA effects of drugs6,7.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>CaCl2</td>
			<td>MECKLIN</td>
			<td>C805225</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Biomed</td>
			<td>SH5001-01</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>HARVEYBIO</td>
			<td>MB5051</td>
			<td></td>
		</tr>
		<tr>
			<td>Small animal ventilator</td>
			<td>RWD</td>
			<td>H1550501-012</td>
			<td></td>
		</tr>
	</tbody>
</table>

a calcium phosphate-induced mouse abdominal aortic aneurysm model

An abdominal aortic aneurysm (AAA) is a life-threatening cardiovascular disease that occurs worldwide and is characterized by irreversible dilation of the abdominal aorta. Currently, several chemically induced murine AAA models are used, each simulating a different aspect of the pathogenesis of AAA. The calcium phosphate-induced AAA model is a rapid and cost-effective model compared to the angiotensin II- and elastase-induced AAA models. The application of CaPO4 crystals to the mouse aorta results in elastic fiber degradation, loss of smooth muscle cells, inflammation, and calcium deposition associated with aortic dilation. This article introduces a standard protocol for the CaPO4-induced AAA model. The protocol includes material preparation, the surgical application of the CaPO4&#160;to the adventitia of the infrarenal abdominal aorta, the harvesting of aortas to visualize aortic aneurysms, and histological analyses in mice.

14 days after CaPO4 application, the C57BL/6J male mice were euthanized, and their aortas were harvested and cleaned. The morphology of the aortas was imaged to visualize AAA formation. As shown in Figure 1A-B, the application of CaPO4 led to dilation of the infrarenal abdominal aorta. Histologically, CaPO4 resulted in a dramatic degradation of elastic fibers, as illustrated by elastin breaks (Figure 1C</...

Watch this Scientific Journal Video about A Calcium Phosphate-Induced Mouse Abdominal Aortic Aneurysm Model at JoVE.com

A Calcium Phosphate-Induced Mouse Abdominal Aortic Aneurysm Model

This protocol describes a calcium phosphate-induced abdominal aortic aneurysm (AAA) mouse model to study the pathological features and molecular mechanisms of AAAs.

An abdominal aortic aneurysm (AAA) is a life-threatening cardiovascular disease that occurs worldwide and is characterized by irreversible dilation of the ...

This protocol describes the calcium phosphate induced abdominal aortic aneurysm triple A mouse model to study the pathological features in the molecular mechanisms of abdominal aortic aneurysm. The calcium phosphate induced abdominal aortic aneurysm model is a rapid and the cost-effective model compared to the angiotensin tube and the elastase induced abdominal aortic aneurysm models. Demonstrating the procedure will be Zeyu Cai, post-doctor here, a fellow from our laboratory.To begin cut 0.3 centimeter wide strips of powder free rubber gloves and gauze. Then purchase eight to ten week old C57BL6J Mayo mice, and house the animals in an air conditioned environment with a 12 hour light dark cycle and free access to food and water. Next, autoclave the gauze, cotton swabs, scissors, and forceps prior to surgery, and obtain Betadine, 70%ethanol, and antiseptic hand wash.Shave the abdominal hair of the anesthetized mouse using an electric clipper or hair removal cream. Then swab and wipe the shaved area with Betadine, followed by 70%ethanol several times in a circular motion. Then make an approximately 1.5 centimeter incision at the lower abdomen along the midline of the abdomen using scissors.Using a sterile cotton swab moistened with normal saline, carefully remove the intestine until the infrarenal aorta is visible. Next, dissect the connective tissue and fat from the infrarenal aorta for an approximately 0.5 centimeter section while avoiding any tearing in the small vessels to the dorsal side. Then pack a piece of the saline soaked rubber glove strip under the abdominal aorta and abdominal main vein while wiping off excess liquid using a cotton swab.Afterward, pack a piece of gauze soaked with 0.5 molar calcium chloride on the adventitia of the infrarenal abdominal vasculature for 10 minutes. Remove the gauze and pack another piece of gauze soaked with PBS solution for five minutes to generate calcium ortho phosphate crystals in C2 in the adventitia of the aorta. Then carefully remove the rubber glove strip and gauze.After resetting the intestinal tract of the mouse, suture the abdominal incision and skin with a five zero suture. Next, place the mouse on a heating pad until the mouse regains consciousness. House the mouse for another 14 days and monitor the mouse closely after the surgery and observe at least every day subsequently.After 14 days of the surgery, cut open the euthanized mouse thoracic and abdominal cavities ventrally and cut open the right atrium. Profuse the mice with PBS buffer through the left ventricle of the heart to remove blood in the aorta and then perfuse with 4%paraformaldehyde as described in the manuscript. Remove adventitial tissue and fat carefully under a stereo microscope.Then harvest the aorta and place it in a tube containing five milliliters of 4%paraformaldehyde for 48 hours. Then pin the aorta on a black wax plate with insect needles and proceed to acquire aortic images. After 14 days of calcium orthophosphate application the morphology of the aortas was imaged to visualize abdominal aortic aneurysm formation.It was observed that calcium orthophosphate led to dilation of the infrarenal abdominal aorta and resulted in a dramatic degradation of elastic fibers as illustrated by elastin breaks. Using a sterile cotton swab moistened with normal saline to remove the intestine until the infrarenal aorta is visible and then extracting the connective tissue and the fat from the infrarenal aorta. Overall, the calcium phosphorate induced mice abdominal aortic aneurysm model is a rapid and cost effective approach to explore molecular mechanisms and the therapeutic strategies of abdominal aortic aneurysm and could be applied in parallel with the other models to fully imitate the features of human abdominal aortic aneurysm.

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3.7K visualizações.  Peking University. Este protocolo descreve o modelo de camundongo triplo A do aneurisma da aorta abdominal induzido por fosfato de cálcio para estudar as características patológicas nos mecanismos moleculares do aneurisma da aorta abdominal. O modelo de aneurisma da aorta abdominal induzido por fosfato de cálcio é um modelo rápido e custo-efetivo em comparação com os modelos de tubo de angiotensina e aneurisma da aorta abdominal induzido por elastase. Demonstrando o procedimento estará Zeyu Cai, pós-m...