We thank the Unit for Laboratory Animal Medicine of the University of Michigan for their help with animal feeding and breeding. This study is supported by NIH RO1 HL138139, NIH RO1 HL153710 to&#160;J. Zhang, NIH RO1 HL109946, RO1 HL134569 to Y.E. Chen, and the American Heart Association grant 20POST35110064 to G. Zhao.

The animal protocols were approved by the University of Michigan Institutional Animal Care and Use Committee (PRO00010092). Male and female C57BL/6J wild-type (WT) mice, ~7 weeks of age, were used for the experiments.
1. Animal preparation
<ol>
	<li>Feed the mice with the standard chow diet (see Table of Materials) before and after the surgery. 
	&#8203;NOTE: Different strains and ages of mice can be used. However, age ranging from 5.5-12 week-old is recommended to achieve maximal incidence rate.</li>
	<li>For each mouse, administer 5 mg/kg of Carprofen subcutaneously 30 min before the induction of anesthesia.</li>
	<li>After 30 min, administer 100 mg/kg of Ketamine and 5mg/kg of Xylazine via intraperitoneal injection to induce anesthesia.</li>
</ol>
2. Preparation for surgery
<ol>
	<li>Prepare the surgical material.
	<ol>
		<li>Cut the nitrile gloves into strips of 4 cm x 4 mm. Cut the cotton pads into pieces of 3 cm x 2 mm. Autoclave these with other surgical instruments, including surgical scissors, tissue forceps, and Halsted-Mosquito hemostats (see Table of Materials).</li>
	</ol>
	</li>
	<li>Place the mouse in the supine position on a sterile absorbent dressing pad. Immobilize the front and hind paws with surgical tape.</li>
	<li>Use cotton-tipped applicators (see Table of Materials) to brush hair remover lotion over the middle and lower abdominal area, then swab the area with surgical gauze to remove the hair.</li>
	<li>Disinfect the surgical area&#160;at least three times in a circular motion with alternating applications of 70% alcohol and an iodine-based or chlorhexidine-based scrub. Allow to dry.</li>
</ol>
3. Surgery procedure
<ol>
	<li>Perform the following steps to access the abdominal cavity.
	<ol>
		<li>Test the mouse for the lack of toe-pinch response before skin incision.</li>
		<li>Make a 2.5 cm longitudinal incision on the skin along the midline of the middle and lower abdomen using surgical scissors.</li>
		<li>Gently pull up the underlying muscle and make a 2.5 cm longitudinal incision along linea alba to access the abdominal cavity.</li>
	</ol>
	</li>
	<li>Expose the abdominal aorta.
	<ol>
		<li>Use wetted cotton-tipped applicators to move the intestines and stomach to the right side of the mouse. 
		NOTE: Ideally, this will expose the infrarenal segment of the aorta. If the aorta is hard to locate, the right kidney and right renal artery can identify the aorta (as the right kidney has a slightly lower anatomical location than the left kidney).</li>
		<li>Use forceps to gently remove the connective tissue covering the abdominal aorta and inferior vena cava (IVC). 
		NOTE: The abdominal aorta and IVC are contained within the same vessel sheath. It is unnecessary to remove all the connective tissue as complete removal would increase the risk of damaging these two vessels.</li>
		<li>Use forceps to gently dissect the backside of the abdominal aorta and IVC from the underlying muscles. 
		NOTE: Tips of the forceps should go into the backside of the sheath transversely and create a hole in the fascia that connects the sheath to the underlying muscles. Once the hole is made, extend its size by slowly releasing the forceps.</li>
		<li>Place a piece of the 4 cm x 4 mm glove stripe (as mentioned earlier, step 2.1.1) through the backside of the abdominal aorta and IVC, then straighten the stripe. Place the stripe ~0.5 cm away from the right renal artery. 
		NOTE: Ensure that the hole is big enough so that the surrounding fascia will not twist the stripe.</li>
		<li>Above the stripe, place a piece of 3 cm x 2 mm cotton pad through the backside of the abdominal aorta and IVC, then straighten the cotton pad.</li>
	</ol>
	</li>
	<li>Incubate the elastase.
	<ol>
		<li>Use a pipette to drop 30 &#181;L of porcine pancreatic elastase (total enzymatic activity of 1.8 unit, see Table of Materials) onto the aorta segment above the cotton pad, then wrap the cotton pad and stripe around the aorta and IVC. Rinse a piece of 10 cm x 10 cm gauze with sterile 0.9% saline and place it on the abdomen. 
		&#8203;NOTE: The gauze needs only partial rinsing as overdoing would risk diluting the elastase beneath.</li>
		<li>After 30 min, remove the stripe and cotton pad with forceps.</li>
	</ol>
	</li>
	<li>Close the abdominal cavity following the steps below.
	<ol>
		<li>Irrigate the aorta and abdominal cavity with 500 &#181;L of sterile 0.9% saline. Use a 10 cm x 10 cm gauze to absorb the remaining saline.</li>
		<li>Reapproximate the muscle layers with a running 6-0 non-absorbable monofilament suture.</li>
		<li>Close skin with 3-4 interrupted 6-0 non-absorbable monofilament sutures.</li>
	</ol>
	</li>
</ol>
4. Post-operative care
<ol>
	<li>Administer 5mg/kg of Carprofen subcutaneously on the post-operative Day 1.</li>
	<li>Remove skin sutures on the post-operative Day 10.</li>
</ol>
5. Measurement of abdominal aorta aneurysm diameter
<ol>
	<li>Euthanize the mice by CO2 overdosing on post-operative Day 14. This represents the time point of maximal dilatation.</li>
	<li>Access the abdominal cavity as described in step 3.1.</li>
	<li>Perform vascular perfusion by injecting 10 mL of 0.9% saline into the circulation through the left ventricle.</li>
	<li>Expose the infrarenal segment of the abdominal aorta as described in steps 3.1-3.2. Carefully remove the surrounding connective tissue and separate the abdominal aorta from IVC.</li>
	<li>Measure the diameter of the abdominal aorta with a caliper.</li>
</ol>

<ol>
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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=Ruptured+abdominal+aortic+aneurysms:+The+excessive+mortality+rate+of+conventional+repair.">Ruptured abdominal aortic aneurysms: The excessive mortality rate of conventional repair.</a> Journal of Vascular Surgery. 34 (1), 41-46 (2001).</li><li>Lederle, F. 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=Rupture+rate+of+large+abdominal+aortic+aneurysms+in+patients+refusing+or+unfit+for+elective+repair.">Rupture rate of large abdominal aortic aneurysms in patients refusing or unfit for elective repair.</a> JAMA. 287 (22), 2968-2972 (2002).</li><li>Kochanek, K. D., Xu, J., Murphy, S. L., Minino, A. M., Kung, H. 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S. <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+used+to+explore+abdominal+aortic+aneurysms:+A+systematic+review.">Animal models used to explore abdominal aortic aneurysms: A systematic review.</a> European Journal of Vascular and Endovascular Surgery. 52 (4), 487-499 (2016).</li><li>Tsui, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+models+of+abdominal+aortic+aneurysms.">Experimental models of abdominal aortic aneurysms.</a> Open Cardiovascular Medicine Journal. 4, 221-230 (2010).</li><li>Manning, M. W., Cassi, L. A., Huang, J., Szilvassy, S. J., Daugherty, A. <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:+Fresh+insights+from+a+novel+animal+model+of+the+disease.">Abdominal aortic aneurysms: Fresh insights from a novel animal model of the disease.</a> Vascular Medicine. 7 (1), 45-54 (2002).</li><li>Chiou, A. C., Chiu, B., Pearce, W. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Murine+aortic+aneurysm+produced+by+periarterial+application+of+calcium+chloride.">Murine aortic aneurysm produced by periarterial application of calcium chloride.</a> Journal of Surgical Research. 99 (2), 371-376 (2001).</li><li>Allaire, E., Guettier, C., Bruneval, P., Plissonnier, D., Michel, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-free+arterial+grafts:+Morphologic+characteristics+of+aortic+isografts,+allografts,+and+xenografts+in+rats.">Cell-free arterial grafts: Morphologic characteristics of aortic isografts, allografts, and xenografts in rats.</a> Journal of Vascular Surgery. 19 (3), 446-456 (1994).</li><li>Anidjar, S., 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=Elastase-induced+experimental+aneurysms+in+rats.">Elastase-induced experimental aneurysms in rats.</a> Circulation. 82 (3), 973-981 (1990).</li><li>Sun, J., 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=Mast+cells+modulate+the+pathogenesis+of+elastase-induced+abdominal+aortic+aneurysms+in+mice.">Mast cells modulate the pathogenesis of elastase-induced abdominal aortic aneurysms in mice.</a> Journal of Clinical Investigation. 117 (11), 3359-3368 (2007).</li><li>Bhamidipati, C. 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=Development+of+a+novel+murine+model+of+aortic+aneurysms+using+peri-adventitial+elastase.">Development of a novel murine model of aortic aneurysms using peri-adventitial elastase.</a> Surgery. 152 (2), 238-246 (2012).</li><li>Pyo, R., 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=Targeted+gene+disruption+of+matrix+metalloproteinase-9+(gelatinase+B)+suppresses+development+of+experimental+abdominal+aortic+aneurysms.">Targeted gene disruption of matrix metalloproteinase-9 (gelatinase B) suppresses development of experimental abdominal aortic aneurysms.</a> Journal of Clinical Investigation. 105 (11), 1641-1649 (2000).</li><li>Tanaka, A., Hasegawa, T., Chen, Z., Okita, Y., Okada, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+rat+model+of+abdominal+aortic+aneurysm+using+a+combination+of+intraluminal+elastase+infusion+and+extraluminal+calcium+chloride+exposure.">A novel rat model of abdominal aortic aneurysm using a combination of intraluminal elastase infusion and extraluminal calcium chloride exposure.</a> Journal of Vascular Surgery. 50 (6), 1423-1432 (2009).</li><li>Busch, 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=Four+surgical+modifications+to+the+classic+elastase+perfusion+aneurysm+model+enable+haemodynamic+alterations+and+extended+elastase+perfusion.">Four surgical modifications to the classic elastase perfusion aneurysm model enable haemodynamic alterations and extended elastase perfusion.</a> European Journal of Vascular and Endovascular Surgery. 56 (1), 102-109 (2018).</li><li>Liang, W., 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=KLF11+protects+against+venous+thrombosis+via+suppressing+tissue+factor+expression.">KLF11 protects against venous thrombosis via suppressing tissue factor expression.</a> Thrombosis and Haemostasis. , (2021).</li><li>Marinov, G. 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The authors have nothing to disclose.

The elastase-induced AAA model was first reported by Anidjar et al. using rats in 199017. A variety of modified versions have been introduced in the past thirty years, along with significant improvement in the surgical techniques19,20,21,22. Hundreds of institutes use elastase-induced AAA models as the second most used rodent experimental model for AAA studies<sup class=...

Abdominal aortic aneurysm (AAA) is defined as a segmental dilatation of the abdominal aorta with at least a 50% increase of vessel diameter1. AAA is potentially fatal, as the rupture can result in an extremely high mortality rate, even with intervention2,3,4. It has been reported that AAA is responsible for approximately 13,000 deaths annually in the USA, which makes it the 10th leading cause of death1,5.
The pathogenesis of AAA is not yet wholly understood6,7,8. To investigate the molecular mechanism of AAA and test potential therapeutic targets, several experimental AAA models have been established9,10. Rodent models of AAA include elastase, calcium chloride, angiotensin II and xenograft models, among which the elastase-induced AAA model is the second most used model10,11,12,13,14,15,16,17. This model involves direct infusion or application of porcine pancreatic elastase (PPE) to the infrarenal segment of the aorta. The penetration of elastase into the medial layer of the aorta will cause degradation of elastic lamellae and infiltration of inflammatory cells, leading to the loss of aortic wall integrity and subsequent dilation of abdominal aorta7,18. The elastase-induced AAA model was first reported by Anidjar et al. in 1990 using rats, in which an isolated segment of the aorta was perfused with elastase17. Later in 2012, a modified model using a periadventitial application of PPE was reported by Bhamidipati et al.19. Nowadays, most surgeries for the elastase-induced AAA model are inspired by Bhamidipati&#39;s group and are performed with the external application rather than intraluminal perfusion of PPE. Although the external application has less requirement on fine surgical skills, the incidence rate of AAA is relatively lower and size somewhat smaller than that of intraluminal perfusion11,19.
Although widely used in AAA studies, the elastase-induced AAA model possesses certain limitations. One caveat of this model is the inevitable variations of how the surgery is performed, which can lead to the issue of reproducibility. For example, the difference may exist in the surgical procedure regarding how the infrarenal segment of the aorta is isolated and which part of the segment is selected for PPE application among different laboratories. The enzymatic activity of PPE and the time duration of PPE incubation can also vary. These, however, are all essential determinants that affect the eventual AAA formation rate and aneurysm diameter.The variation of these critical determinants makes data comparison of AAA studies from different groups using this model very difficult. Therefore, a standardized surgical procedure is needed as a tool to get comparable results from various institutions.
This article describes a standardized surgical protocol for the elastase-induced AAA model through direct application of PPE to the adventitia of infrarenal abdominal aorta in mice. Details about surgical material and procedures essential for successful and robust generation of AAA in mice using this model will also be discussed.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>6-0 non-absorbable monofilament suture</td>
			<td>Pro Advantage</td>
			<td>P420697</td>
			<td></td>
		</tr>
		<tr>
			<td>Carprofen</td>
			<td>Zoetis Inc.</td>
			<td>NDC: 54771-8507</td>
			<td></td>
		</tr>
		<tr>
			<td>Chow Diet</td>
			<td>LabDiet</td>
			<td>3005659-220</td>
			<td>PicoLab 5L0D</td>
		</tr>
		<tr>
			<td>Cotton Applicator</td>
			<td>Dynarex</td>
			<td>4303</td>
			<td></td>
		</tr>
		<tr>
			<td>Cotton Pad</td>
			<td>Rael</td>
			<td>UPC: 810027130969</td>
			<td></td>
		</tr>
		<tr>
			<td>GraphPad Prism 8</td>
			<td>GraphPad Software Inc.</td>
			<td></td>
			<td>Version 8.4.3</td>
		</tr>
		<tr>
			<td>Grarfe Forceps</td>
			<td>Fine Science Tools</td>
			<td>11051-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Halsted Mosquito Hemostats</td>
			<td>Fine Science Tools</td>
			<td>13009-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Ketamine</td>
			<td>Par Pharmaceutical</td>
			<td>NDC: 42023-0115-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Nitrile gloves</td>
			<td>Fisherbrand</td>
			<td>19-130-1597</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Thermo Fisher</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Porcine pancreatic elastase</td>
			<td>Sigma-Aldrich</td>
			<td>E1250-100MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Scissors</td>
			<td>Fine Science Tools</td>
			<td>14068-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile 0.9% saline solution</td>
			<td>Baxter</td>
			<td>2B1324X</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>Akorn</td>
			<td>NDC: 59399-110-20</td>
			<td></td>
		</tr>
	</tbody>
</table>

mouse abdominal aortic aneurysm model induced by perivascular application of elastase

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

A total of twenty-three 7-week-old wild-type (WT) mice, including 12 females and 11 males, were operated following the presented protocol. The survival rate was 100% (surgical mortality excluded). Maximal abdominal aorta diameter was measured by a caliper.
AAA was defined as dilating the abdominal aorta with a 50% vessel diameter increase. Therefore, a 50% increase in the maximal abdominal aorta diameter was selected as the cut-off point for successful AAA induction. Based upon this criterion,...

Watch this Scientific Journal Video about Mouse Abdominal Aortic Aneurysm Model Induced by Perivascular Application of Elastase at JoVE.com

Mouse Abdominal Aortic Aneurysm Model Induced by Perivascular Application of Elastase

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

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

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5.1K Views.  University of Michigan, Ann Arbor. The present protocol describes a standardized surgical method for the elastase-induced AAA model through the direct application of elastase to the adventitia of infrarenal abdominal aorta in mice. 

Video: Mouse Abdominal Aortic Aneurysm Model Induced by Perivascular Application of Elastase

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 ...

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 ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution2. Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types2. This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications.
The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC)3-5. Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage6. Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line.
Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC1. These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs3,4,7 and higher than that reported for most other iPSC lines8-12. These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines13-15. Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all- iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods.

Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

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 ...

Differentiation Capacity of Human Aortic Perivascular Adipose Progenitor Cells

Adipose tissue is a rich source of multi-potent mesenchymal stem cells (MSC) capable of differentiating into osteogenic, adipogenic, and chondrogenic lineages. Adipogenic differentiation of progenitor cells is a major mechanism driving adipose tissue expansion and dysfunction in response to obesity. Understanding changes to perivascular adipose tissue (PVAT) is thus clinically relevant in metabolic disease. However, previous studies have been predominately performed in the mouse and other animal models. This protocol uses human thoracic PVAT samples collected from patients undergoing coronary artery bypass graft surgery. Adipose tissue from the ascending aorta was collected and used for explantation of the stromal vascular fraction. We previously confirmed the presence of adipose progenitor cells in human PVAT with the capacity to differentiate into lipid-containing adipocytes. In this study, we further analyzed the differentiation potential of cells from the stromal vascular fraction, presumably containing multi-potent progenitor cells. We compared PVAT-derived cells to human bone marrow MSC for differentiation into adipogenic, osteogenic, and chondrogenic lineages. Following 14 days of differentiation, specific stains were utilized to detect lipid accumulation in adipocytes (Oil red O), calcific deposits in osteogenic cells (Alizarin Red), or glycosaminoglycans and collagen in chondrogenic cells (Masson&#8217;s Trichrome). While bone marrow MSC efficiently differentiated into all three lineages, PVAT-derived cells had adipogenic and chondrogenic potential, but lacked robust osteogenic potential.

The goal of this protocol is to test the ability of progenitor cells derived from human perivascular adipose tissue to differentiate into multiple cell lineages. Differentiation was compared to mesenchymal stem cells derived from human bone marrow, which is known to differentiate into adipocyte, osteocyte, and chondrocyte lineages.

Adipose tissue is a rich source of multi-potent mesenchymal stem cells (MSC) capable of differentiating into osteogenic, adipogenic, and chondrogenic ...

Assessment of Vascular Tone Responsiveness using Isolated Mesenteric Arteries with a Focus on Modulation by Perivascular Adipose Tissues

Altered vascular tone responsiveness to pathophysiological stimuli contributes to the development of a wide range of cardiovascular and metabolic diseases. Endothelial dysfunction represents a major culprit for the reduced vasodilatation and enhanced vasoconstriction of arteries. Adipose (fat) tissues surrounding the arteries play important roles in the regulation of endothelium-dependent relaxation and/or contraction of the vascular smooth muscle cells. The cross-talks between the endothelium and perivascular adipose tissues can be assessed ex vivo using mounted blood vessels by a wire myography system. However, optimal settings should be established for arteries derived from animals of different species, ages, genetic backgrounds and/or pathophysiological conditions.

The protocol describes the use of wire myography to evaluate the transmural isometric tension of mesenteric arteries isolated from mice, with special consideration of the modulation by factors released from endothelial cells and perivascular adipose tissues.

Altered vascular tone responsiveness to pathophysiological stimuli contributes to the development of a wide range of cardiovascular and metabolic ...

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 ...

Monitoring Dynamic Growth of Retinal Vessels in Oxygen-Induced Retinopathy Mouse Model

Oxygen-induced retinopathy (OIR) is widely used to study abnormal vessel growth in ischemic retinal diseases, including retinopathy of prematurity (ROP), proliferative diabetic retinopathy (PDR), and retinal vein occlusion (RVO). Most OIR studies observe retinal neovascularization at specific time points; however, the dynamic vessel growth in live mice along a time course, which is essential for understanding the OIR-related vessel diseases, has been understudied. Here, we describe a step-by-step protocol for the induction of the OIR mouse model, highlighting the potential pitfalls, and providing an improved method to quickly quantify areas of vaso-obliteration (VO) and neovascularization (NV) using immunofluorescence staining. More importantly, we monitored vessel regrowth in live mice from P15 to P25 by performing fluorescein fundus angiography (FFA) in the OIR mouse model. The application of FFA to the OIR mouse model allows us to observe the remodeling process during vessel regrowth.

This protocol describes a detailed method for the preparation and immunofluorescence staining of mice retinal flat mounts and analysis. The use of fluorescein fundus angiography (FFA) for mice pups and image processing are described in detail as well.

Oxygen-induced retinopathy (OIR) is widely used to study abnormal vessel growth in ischemic retinal diseases, including retinopathy of prematurity (ROP), ...