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
<ol>
	<li>Cut 0.3 cm wide strips of powder-free rubber gloves and gauze.</li>
	<li>Purchase 8-10-week-old C57BL/6J male mice. House the animals in an air-conditioned environment with a 12 h light-dark cycle and free access to food and water.</li>
	<li>Autoclave the gauze, cotton swabs, scissors, and forceps prior to surgery.</li>
	<li>Obtain betadine, 70% ethanol, and antiseptic hand wash.</li>
	<li>Put on a mask, gown, and sterile gloves.</li>
</ol>
2. Surgical procedure
<ol>
	<li>Feed chewable carprofen tablets (5 mg/kg dose) to an 8-10-week-old C57BL/6J mouse 2-4 h before surgery. Then, place the mouse in an induction chamber (206 mm x 210 mm x 140 mm) with isoflurane at a flow rate of 1.5%-2%.
	<ol>
		<li>Monitor the mouse for about 5 min until respiration is visibly slowed. Ensure that the mouse has no response to pain stimulation before surgery.</li>
	</ol>
	</li>
	<li>Apply an ophthalmic ointment to the eyes and provide thermal support with a heating pad or blanket. Confirm the depth of anesthesia with a toe pinch every 15 minutes during the surgical procedure.</li>
	<li>Shave the abdominal hair of the mouse using an electric clipper or hair removal cream. Swab and wipe the shaved area with betadine, followed by 70% ethanol, several times in a circular motion. Change gloves to maintain sterility.</li>
	<li>Use scissors to make a ~1.5 cm incision at the lower abdomen along the midline of the abdomen.</li>
	<li>Use a sterile cotton swab moistened with normal saline to carefully remove the intestine until the infrarenal aorta is visible.</li>
	<li>Dissect the connective tissue and fat from the infrarenal aorta for an approximately 0.5 cm section. Note the small vessels to the dorsal side and avoid tearing them. There is no need to separate the abdominal aorta from the abdominal main vein.</li>
	<li>Pack a piece of the saline-soaked rubber glove strip under the abdominal aorta and abdominal main vein. Use a cotton swab to wipe off excess liquid.</li>
	<li>Pack a piece of gauze soaked with 0.5 M CaCl2 on the adventitia of the infrarenal abdominal vasculature for 10 min. For the sham mouse group, replace the 0.5 M CaCl2 with normal saline.</li>
	<li>Remove the gauze and pack another piece of gauze soaked with PBS solution for 5 min to generate CaPO4 crystals in situ in the adventitia of the aorta.</li>
	<li>Carefully remove the rubber glove strip and gauze. Reset the intestinal tract of the mouse.</li>
	<li>Suture the abdominal incision and skin with a 5-0 suture.</li>
	<li>Place the mouse on a heating pad until the mouse regains consciousness. Provide postsurgical pain recovery housing and analgesia, according to the local animal ethics committee.</li>
	<li>House the mouse for another 14 days. Monitor the mouse closely after the surgery and observe at least 1x every day subsequently. Perform a necropsy immediately if any mice die during this period.</li>
</ol>
3. Harvesting for imaging of aortas
<ol>
	<li>14 days after the surgery, sacrifice the mice using CO2.</li>
	<li>Cut open the mouse thoracic and abdominal cavities ventrally and cut open the right atrium.</li>
	<li>Perfuse 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 previously described8.</li>
	<li>Harvest the aorta under the stereoscope.</li>
	<li>Place harvested aortas in tubes containing 5 mL of 4% paraformaldehyde for 48 h.</li>
	<li>Remove adventitial tissue and fat carefully under the stereoscope and pin the aorta on a black wax plate with insect needles.</li>
	<li>Acquire aortic images.</li>
</ol>
4. Evaluation of the degradation of the elastic fibers
<ol>
	<li>Cut the aortic aneurysm tissues into serial cryosections (7 &#181;m thick).</li>
	<li>Analyze the elastic fibers using a commercial elastic van Gieson (EVG) staining kit according to the manufacturer&#39;s protocol.</li>
	<li>Grade the elastin degradation. Grade 1: &#60;25% degradation; grade 2: 25% to 50% degradation; grade 3: 50% to 75% degradation; or grade 4: &#62;75% degradation.</li>
</ol>

<ol>
	<li>Kent, K. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Abdominal+aortic+aneurysms.">Abdominal aortic aneurysms.</a> The New England Journal of Medicine. 371, 2101-2108 (2014).</li><li>Patelis, N., 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=Animal+models+in+the+research+of+abdominal+aortic+aneurysms+development.">Animal models in the research of abdominal aortic aneurysms development.</a> Physiological Research. 66 (6), 899-915 (2017).</li><li>Gertz, S. D., Kurgan, A., Eisenberg, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aneurysm+of+the+rabbit+common+carotid+artery+induced+by+periarterial+application+of+calcium-chloride+in+vivo.">Aneurysm of the rabbit common carotid artery induced by periarterial application of calcium-chloride in vivo.</a> Journal of Clinical Investigation. 81 (3), 649-656 (1988).</li><li>Yamanouchi, D., 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=Accelerated+aneurysmal+dilation+associated+with+apoptosis+and+inflammation+in+a+newly+developed+calcium+phosphate+rodent+abdominal+aortic+aneurysm+model.">Accelerated aneurysmal dilation associated with apoptosis and inflammation in a newly developed calcium phosphate rodent abdominal aortic aneurysm model.</a> Journal of Vascular Surgery. 56 (2), 455-461 (2012).</li><li>Wang, Y. T., 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=Influence+of+apolipoprotein+E,+age+and+aortic+site+on+calcium+phosphate+induced+abdominal+aortic+aneurysm+in+mice.">Influence of apolipoprotein E, age and aortic site on calcium phosphate induced abdominal aortic aneurysm in mice.</a> Atherosclerosis. 235 (1), 204-212 (2014).</li><li>Zhao, G., 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=Unspliced+xbp1+confers+VSMC+homeostasis+and+prevents+aortic+aneurysm+formation+via+foxo4+interaction.">Unspliced xbp1 confers VSMC homeostasis and prevents aortic aneurysm formation via foxo4 interaction.</a> Circulation Research. 121 (12), 1331-1345 (2017).</li><li>Jia, Y., 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=Targeting+macrophage+TFEB-14-3-3+epsilon+interface+by+naringenin+inhibits+abdominal+aortic+aneurysm.">Targeting macrophage TFEB-14-3-3 epsilon interface by naringenin inhibits abdominal aortic aneurysm.</a> Cell Discovery. 8 (1), 21 (2022).</li><li>Gage, G. 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. F., Fagnano, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Risk+factors+for+abdominal+aortic+aneurysm+in+population-based+studies:+A+systematic+review+and+meta-analysis.">Risk factors for abdominal aortic aneurysm in population-based studies: A systematic review and meta-analysis.</a> International Journal of Environmental Research and Public Health. 15 (12), 2805 (2018).</li><li>Theivacumar, N. S., Stephenson, M. A., Mistry, H., Valenti, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diabetes+mellitus+and+aortic+aneurysm+rupture:+A+favorable+association.">Diabetes mellitus and aortic aneurysm rupture: A favorable association.</a> Vascular and Endovascular Surgery. 48 (1), 45-50 (2014).</li><li>Tanaka, T., Takei, Y., Yamanouchi, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hyperglycemia+suppresses+calcium+phosphate-induced+aneurysm+formation+through+inhibition+of+macrophage+activation.">Hyperglycemia suppresses calcium phosphate-induced aneurysm formation through inhibition of macrophage activation.</a> Journal of the American Heart Association. 5 (3), 003062 (2016).</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> Journal of Visualized Experiments. (103), e53191 (2015).</li><li>Urry, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutral+sites+for+calcium+ion+binding+to+elastin+and+collagen:+A+charge+neutralization+theory+for+calcification+and+its+relationship+to+atherosclerosis.">Neutral sites for calcium ion binding to elastin and collagen: A charge neutralization theory for calcification and its relationship to atherosclerosis.</a> Proceedings of the National Academy of Sciences of the United States of America. 68 (4), 810-814 (1971).</li><li>Li, Z. Q., 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=Runx2+(runt-related+transcription+factor+2)-mediated+microcalcification+is+a+novel+pathological+characteristic+and+potential+mediator+of+abdominal+aortic+aneurysm.">Runx2 (runt-related transcription factor 2)-mediated microcalcification is a novel pathological characteristic and potential mediator of abdominal aortic aneurysm.</a> Arteriosclerosis, Thrombosis, and Vascular Biology. 40 (5), 1352-1369 (2020).</li><li>Kelly, M. J., Igari, K., Yamanouchi, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osteoclast-like+cells+in+aneurysmal+disease+exhibit+an+enhanced+proteolytic+phenotype.">Osteoclast-like cells in aneurysmal disease exhibit an enhanced proteolytic phenotype.</a> International Journal of Molecular Sciences. 20 (19), 4689 (2019).</li></ol>

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.
The risk factors of human AAA mainly include aging, male gender, smoking, hyperlipidemia, hypertension, and atherosclerosis10. Although not systematically studied, hyperlipidemia was also found to predispose mice to AAA expansion when using the CaPO4 model. Unlike humans, aging plays a minor role in AAA formation in the mouse CaPO4 model5. Previous large epidemiological studies have shown that diabetes is an independent negative risk factor of human AAA11. While some data suggest metformin, a diabetic drug, causes this effect, alternatively it is of interest that, in the CaPO4 model, hyperglycemia inhibits aortic dilation by the suppression of macrophage activation12.
Currently, several chemically induced murine AAA models have been established, including elastase incubation, CaPO4 incubation, and subcutaneous AngII perfusion2. Generally, the elastase and CaPO4 models have been performed in 8-10-week-old male wild-type mice, and the AngII model has been performed in hyperlipidemia mice (such as ApoE-/- and LDLR-/- mice) or 5-6-month-old mice to induce AAA13. All the three models phenocopy the major pathological characteristics of human AAA, including elastic fiber degradation and immune cell infiltration. Compared with the other two models, CaPO4 application leads to a rapid and more than 1.5-fold expansion of the aorta 7 days after surgery, associated with dramatic elastin degradation and calcium deposition4. The CaPO4 model induces a fusiform dilation at the infrarenal abdominal aorta, which mimics the human AAA condition, whereas AngII perfusion induces both suprarenal AAA and thoracic aortic aneurysm. Considering the cost of elastase, AngII, and the osmotic mini-pump, it is more cost-effective to perform the CaPO4 model. However, it is fair to state that the CaPO4 model cannot induce AAA features such as mural thrombus formation and aortic rupture, and the model is rapid and, thus, less suitable for performing intervention studies with existing AAA. The CaPO4 model is ideal for working with genetically modified mice to assess susceptibility to AAA development.
The mechanism underlying CaPO4-induced AAA formation has not been fully elucidated yet. Previous studies hint that the calcium ion may directly bind to the major arterial structural components, elastin and collagen, facilitating extracellular matrix degradation and decreased vessel wall stability14. CaPO4 crystals have also been identified to trigger significant NOD-like receptor protein 3 (NLRP3) inflammasome activation and smooth muscle cell apoptosis4,15. Besides, microcalcification crystals are capable of inducing mononuclear cells into osteoclast-like cells and promoting matrix metalloproteinase (MMP) production and may, ultimately, result in aortic expansion16.
When performing the CaPO4 model, several issues must be avoided to improve the success rate. One should avoid tearing the dorsal branch blood vessels, avoid the addition of excess CaCl2 solution into the abdominal cavity, and avoid an overdose of anesthesia. Mice with severe bleeding during surgery or with postoperative infection should be excluded from the experiment. As previously reported, when the number of the observations is sufficient, the maximal diameters of the aortas in the CaPO4 model are usually normally distributed, which is different from the AngII model7. Therefore, we recommend a comparison of the maximal diameters of aortas instead of AAA incidence when using the CaPO4 model.
Overall, the CaPO4-induced mice AAA model is a rapid and cost-effective approach to explore the molecular mechanisms and therapeutic strategies of AAA and could be applied in parallel with the other models to fully imitate the features of human AAA.

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).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64173/64173fig01.jpg" /> 
Figure 1: The 8-week-old male C57BL/6J mice were treated with saline (sham) or CaPO4 harvested after 14 days. (A) Representative images of infrarenal abdominal aorta under a stereoscope. (B) Representative morphology images of aortas from mice; scale bar = 1 mm. (C) Representative images of elastic van Gieson staining of aortas. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64173/64173fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

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3.8K Views.  Peking University. This protocol describes a calcium phosphate-induced abdominal aortic aneurysm (AAA) mouse model to study the pathological features and molecular mechanisms of AAAs.

Video: A Calcium Phosphate-Induced Mouse Abdominal Aortic Aneurysm Model

Creation of Murine Experimental Abdominal Aortic Aneurysms with Elastase 

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

Creation of Murine Experimental Abdominal Aortic Aneurysms with Elastase

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

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

Aortic Ring Assay

Angiogenesis, the sprouting of blood vessels from preexisting vasculature is associated with both natural and pathological processes. Various angiogenesis assays involve the study of individual endothelial cells in culture conditions (1). The aortic ring assay is an angiogenesis model that is based on organ culture. In this assay, angiogenic vessels grow from a segment of the aorta (modified from (2)). Briefly, mouse thoracic aorta is excised, the fat layer and adventitia are removed, and rings approximately 1 mm in length are prepared. Individual rings are then embedded in a small solid dome of basement matrix extract (BME), cast inside individual wells of a 48-well plate. Angiogenic factors and inhibitors of angiogenesis can be directly added to the rings, and a mixed co-culture of aortic rings and other cell types can be employed for the study of paracrine angiogenic effects. Sprouting is observed by inspection under a stereomicroscope over a period of 6-12 days. Due to the large variation caused by the irregularities in the aortic segments, experimentation in 6-plicates is strongly advised. Neovessel outgrowth is monitored throughout the experiment and imaged using phase microscopy, and supernatants are collected for measurement of relevant angiogenic and anti-angiogenic factors, cell death markers and nitrite.

Angiogenesis, the sprouting of blood vessels from pre-existing vasculature, is associated with both natural and pathological processes. Here we demonstrate an aortic ring assay that allows angiogenic potentiators and inhibitors to be directly added to aortic rings in culture. Sprouting and neovessel outgrowth can be determined by inspecting the aortic rings over a period of 6-12 days.

Angiogenesis, the sprouting of blood vessels from preexisting vasculature is associated with both natural and pathological processes. Various angiogenesis ...

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Human cancer and response to therapy is better represented in orthotopic animal models. This paper describes the development of an orthotopic mouse model of ovarian cancer, treatment of cancer via oral delivery of drugs, and monitoring of tumor cell behavior in response to drug treatment in real time using in vivo imaging system. In this orthotopic model, ovarian tumor cells expressing luciferase are applied topically by injecting them directly into the mouse bursa where each ovary is enclosed. Upon injection of D-luciferin, a substrate of firefly luciferase, luciferase-expressing cells generate bioluminescence signals. This signal is detected by the in vivo imaging system and allows for a non-invasive means of monitoring tumor growth, distribution, and regression in individual animals. Drug administration via oral gavage allows for a maximum dosing volume of 10 mL/kg body weight to be delivered directly to the stomach and closely resembles delivery of drugs in clinical treatments. Therefore, techniques described here, development of an orthotopic mouse model of ovarian cancer, oral delivery of drugs, and in vivo imaging, are useful for better understanding of human ovarian cancer and treatment and will improve targeting this disease.

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Orthotopic animal models of ovarian cancer replicate better human disease and therefore enhance our understanding of cancer progression and tumor response to therapy. A mouse model receives an intrabursal injection of luciferase-expressing ovarian tumor cells. Treatment is administered via oral gavage. Tumor growth is monitored by in vivo imaging system.

Human cancer and response to therapy is better represented in orthotopic animal models.  This paper describes the development of an orthotopic mouse model ...

Isolation and Primary Culture of Mouse Aortic Endothelial Cells

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an important model for cardiovascular disease research. This study demonstrates a simple method to isolate and culture endothelial cells from the mouse aorta without any special equipment. To isolate endothelial cells, the thoracic aorta is quickly removed from the mouse body, and the attached adipose tissue and connective tissue are removed from the aorta. The aorta is cut into 1 mm rings. Each aortic ring is opened and seeded onto a growth factor reduced matrix with the endothelium facing down. The segments are cultured in endothelial cell growth medium for about 4 days. The endothelial sprouting starts as early as day 2. The segments are then removed and the cells are cultured continually until they reach confluence. The endothelial cells are harvested using neutral proteinase and cultured in endothelial cell growth medium for another two passages before being used for experiments. Immunofluorescence staining indicated that after the second passage the majority of cells were double positive for Dil-ac-LDL uptake, Lectin binding, and CD31 staining, the typical characteristics of endothelial cells. It is suggested that cells at the second to third passages are suitable for in vitro and in vivo experiments to study the endothelial biology. Our protocol provides an effective means of identifying specific cellular and molecular mechanisms in endothelial cell physiopathology.

The vascular endothelial cells play a significant role in many important cardiovascular disorders. This article describes a simple method to isolate and expand endothelial cells from the mouse aorta without using any special equipment. Our protocol provides an effective means of identifying mechanisms in endothelial cell physiopathology.

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an ...

Trans-inner Cell Mass Injection of Embryonic Stem Cells Leads to Higher Chimerism Rates

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current blastocyst injection protocol. Here we report that a simple rotation of the embryo, and injection through Trans-Inner cell mass (TICM) increased the percentage of chimeric mice from 31% to 50%, with no additional equipment or further specialized training. 26 different inbred clones, and 35 total clones were injected over a period of 9 months. There was no significant difference in either pregnancy rate or recovery rate of embryos between traditional injection techniques and TICM. Therefore, without any major alteration in the injection process and a simple positioning of the blastocyst and injecting through the ICM, releasing the ES cells into the blastocoel cavity can potentially improve the quantity of chimeric production and subsequent germline transmission.

Here we present a protocol to increase chimera production without the use of new equipment. A simple orientation change of the embryo for injection can increase the number of embryos produced, and potentially reduce the timeline to germline transmission.

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current ...

A Mouse Model of Intestinal Partial Obstruction

Intestinal obstructions, that impede or block peristaltic movement, can be caused by abdominal adhesions and most gastrointestinal (GI) diseases including tumorous growths. However, the cellular remodeling mechanisms involved in, and caused by, intestinal obstructions are poorly understood. Several animal models of intestinal obstructions have been developed, but the mouse model is the most cost/time effective. The mouse model uses the surgical implantation of an intestinal partial obstruction (PO) that has a high mortality rate if it is not performed correctly. In addition, mice receiving PO surgery fail to develop hypertrophy if an appropriate blockade is not used or not properly placed. Here, we describe a detailed protocol for PO surgery which produces reliable and reproducible intestinal obstructions with a very low mortality rate. This protocol utilizes a surgically placed silicone ring that surrounds the ileum which partially blocks digestive movement in the small intestine. The partial blockage makes the intestine become dilated due to the halt of digestive movement. The dilation of the intestine induces smooth muscle hypertrophy on the oral side of the ring that progressively develops over 2 weeks until it causes death. The surgical PO mouse model offers an in vivo model of hypertrophic intestinal tissue useful for studying pathological changes of intestinal cells including smooth muscle cells (SMC), interstitial cells of Cajal (ICC), PDGFR&#945;+, and neuronal cells during the development of intestinal obstruction.

Intestinal obstructions are a partial or complete blockage of the intestine that can cause severe abdominal pain, nausea, vomiting, and preventing the passage of stool. This procedure for creating intestinal partial obsructions in mice is reliable in studying the mechanisms underlying pathological cell growth and death in the intestine.

Intestinal obstructions, that impede or block peristaltic movement, can be caused by abdominal adhesions and most gastrointestinal (GI) diseases including ...

Simultaneous Measurements of Intracellular Calcium and Membrane Potential in Freshly Isolated and Intact Mouse Cerebral Endothelium

Cerebral arteries and their respective microcirculation deliver oxygen and nutrients to the brain via blood flow regulation. Endothelial cells line the lumen of blood vessels and command changes in vascular diameter as needed to meet the metabolic demand of neurons. Primary endothelial-dependent signaling pathways of hyperpolarization of membrane potential (Vm) and nitric oxide typically operate in parallel to mediate vasodilation and thereby increase blood flow. Although integral to coordinating vasodilation over several millimeters of vascular length, components of endothelium-derived hyperpolarization (EDH) have been historically difficult to measure. These components of EDH entail intracellular Ca2+ [Ca2+]i increases and subsequent activation of small- and intermediate conductance Ca2+-activated K+ (SKCa/IKCa) channels.
Here, we present a simplified illustration of the isolation of fresh endothelium from mouse cerebral arteries; simultaneous measurements of endothelial [Ca2+]i and Vm using Fura-2 photometry and intracellular sharp electrodes, respectively; and a continuous superfusion of salt solutions and pharmacological agents under physiological conditions (pH 7.4, 37 &#176;C). Posterior cerebral arteries from the Circle of Willis are removed free of the posterior communicating and the basilar arteries. Enzymatic digestion of cleaned posterior cerebral arterial segments and subsequent trituration facilitates removal of adventitia, perivascular nerves, and smooth muscle cells. Resulting posterior cerebral arterial endothelial &#34;tubes&#34; are then secured under a microscope and examined using a camera, photomultiplier tube, and one to two electrometers while under continuous superfusion. Collectively, this method can simultaneously measure changes in endothelial [Ca2+]i and Vm in discrete cellular locations, in addition to the spreading of EDH through gap junctions up to millimeter distances along the intact endothelium. This method is expected to yield a high-throughput analysis of the cerebral endothelial functions underlying mechanisms of blood flow regulation in the normal and diseased brain.

Demonstrated here are protocols for (1) freshly isolating intact cerebral endothelial "tubes" and (2) simultaneous measurements of endothelial calcium and membrane potential during endothelium-derived hyperpolarization. Further, these methods allow for pharmacological tuning of endothelial cell calcium and electrical signaling as individual or interactive experimental variables.

Cerebral arteries and their respective microcirculation deliver oxygen and nutrients to the brain via blood flow regulation. Endothelial cells line the ...

Imaging Calcium Dynamics in Subpopulations of Mouse Pancreatic Islet Cells

Pancreatic islet hormones regulate blood glucose homeostasis. Changes in blood glucose induce oscillations of cytosolic calcium in pancreatic islet cells that trigger secretion of three main hormones: insulin (from &#946;-cells), glucagon (&#945;-cells) and somatostatin (&#948;-cells). &#946;-Cells, which make up the majority of islet cells and are electrically coupled to each other, respond to the glucose stimulus as one single entity. The excitability of the minor subpopulations, &#945;-cells and &#948;-cells (making up around 20% (30%) and 4% (10%) of the total rodent1 (human2) islet cell numbers, respectively) is less predictable and is therefore of special interest.
Calcium sensors are delivered into the peripheral layer of cells within the isolated islet. The islet or a group of islets is then immobilized and imaged using a fluorescence microscope. The choice of the imaging mode is between higher throughput (wide-field) and better spatial resolution (confocal). Conventionally, laser scanning confocal microscopy is used for imaging tissue, as it provides the best separation of the signal between the neighboring cells. A wide-field system can be utilized too, if the contaminating signal from the dominating population of &#946;-cells is minimized.
Once calcium dynamics in response to specific stimuli have been recorded, data are expressed in numerical form as fluorescence intensity vs. time, normalized to the initial fluorescence and baseline-corrected, to remove the effects linked to bleaching of the fluorophore. Changes in the spike frequency or partial area under the curve (pAUC) are computed vs. time, to quantify the observed effects. pAUC is more sensitive and quite robust whereas spiking frequency provides more information on the mechanism of calcium increase.
Minor cell subpopulations can be identified using functional responses to marker compounds, such as adrenaline and ghrelin, that induce changes in cytosolic calcium in a specific populations of islet cells.

Here, we present a protocol for imaging and quantifying calcium dynamics in heterogeneous cell populations, such as pancreatic islet cells. Fluorescent reporters are delivered into the peripheral layer of cells within the islet, which is then immobilized and imaged, and per-cell analysis of the dynamics of fluorescence intensity is performed.

Pancreatic islet hormones regulate blood glucose homeostasis. Changes in blood glucose induce oscillations of cytosolic calcium in pancreatic islet cells ...

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A)

Studying the structure and the dynamics of kinetochores and centromeres is important in understanding chromosomal instability (CIN) and cancer progression. How the chromosomal location and function of a centromere (i.e., centromere identity) are determined and participate in accurate chromosome segregation is a fundamental question. CENP-A is proposed to be the non-DNA indicator (epigenetic mark) of centromere identity, and CENP-A ubiquitylation is required for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability.
Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-CENP-A K124R mutant suggesting that ubiquitylation at a different lysine is induced because of the EYFP tagging in the CENP-A K124R mutant protein. Lysine 306 (K306) ubiquitylation in EYFP-CENP-A K124R was successfully identified, which corresponds to lysine 56 (K56) in CENP-A through mass spectrometry analysis. A caveat is discussed in the use of GFP/EYFP or the tagging of high molecular weight protein as a tool to analyze the function of a protein. Current technical limit is also discussed for the detection of ubiquitylated bands, identification of site-specific ubiquitylation(s), and visualization of ubiquitylation in living cells or a specific single cell during the whole cell cycle.
The method of mass spectrometry analysis presented here can be applied to human CENP-A protein with different tags and other centromere-kinetochore proteins. These combinatory methods consisting of several assays/analyses could be recommended for researchers who are interested in identifying functional roles of ubiquitylation.

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A EYFP-CENP-A

CENP-A ubiquitylation is an important requirement for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability. Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A) protein.

Studying the structure and the dynamics of kinetochores and centromeres is important in understanding chromosomal instability (CIN) and cancer ...

A Mouse Model of Lumbar Spine Instability

Intervertebral disc degeneration (IDD) is a common pathological change leading to low back pain. Appropriate animal models are desired for understanding the pathological processes and evaluating new drugs. Here, we introduced a surgically induced lumbar spine instability (LSI) mouse model that develops IDD starting from 1 week post operation. In detail, the mouse under anesthesia was operated by low back skin incision, L3&#8211;L5 spinous processes exposure, detachment of paraspinous muscles, resection of processes and ligaments, and skin closure. L4&#8211;L5 IVDs were chosen for the observation. The LSI model develops lumbar IDD by porosity and hypertrophy in endplates at an early stage, decrease in intervertebral disc volume, shrinkage in nucleus pulposus at an intermediate stage, and bone loss in lumbar vertebrae (L5) at a later stage. The LSI mouse model has the advantages of strong operability, no requirement of special equipment, reproducibility, inexpensive, and relatively short period of IDD development. However, LSI operation is still a trauma that causes inflammation within the first week post operation. Thus, this animal model is suitable for study of lumbar IDD.

We developed a lumbar intervertebral disc degeneration mouse model by resection of L3&#8211;L5 spinous processes along with supra- and inter-spinous ligaments and detachment of paraspinous muscles.

Intervertebral disc degeneration (IDD) is a common pathological change leading to low back pain. Appropriate animal models are desired for understanding ...

A Calcium Phosphate-Induced Mouse Abdominal Aortic Aneurysm Model

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Simultaneous Measurements of Intracellular Calcium and Membrane Potential in Freshly Isolated and Intact Mouse Cerebral Endothelium

Imaging Calcium Dynamics in Subpopulations of Mouse Pancreatic Islet Cells

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A)

A Mouse Model of Lumbar Spine Instability