We thank Hsia Ru-ching PhD, from the Electron Microscopy Core Facility of University of Maryland School of Medicine, for her technical support in processing the scanning electronic microscopy samples.

Ethics Statement: The protocols for animal handling were approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Maryland (protocol number 0416009) and conducted according to AAALAC-International standards.
1. Surgical Procedure
<ol>
	<li>Anesthetic Technique
	<ol>
		<li>Sterilize all instruments used in survival surgery with steam sterilization at 121 &#176;C for 30 min.</li>
		<li>Induce anesthesia via an induction tank with 100% O2 and 2.5% isoflurane delivered via precision vaporizer. Post-induction, discontinue the isoflurane and flush the chamber with O2. Maintain anesthesia with 1.5-2% isoflurane via face mask and 1 L/min O2 by inhalation.</li>
		<li>Attach both the induction chamber and the face mask to a charcoal scavenger for waste gas adsorption to protect personnel. Ensure an adequate anesthetic plane by demonstrating that there is no response to noxious stimuli (toe pinch).</li>
		<li>Create an operative field consisting of a surgical tray with isothermal pad to provide thermal support during surgery. An additional isothermal pad will provide thermal support to animals in their recovery cage.</li>
	</ol>
	</li>
	<li>Animal Preparation
	<ol>
		<li>Carry out the following investigation on 10-12 weeks old male C57BL/6 mice.</li>
		<li>Remove the hair on the ventral abdominal surface of the animal from the sternum to the inguinal region with a depilatory agent or an electrical clipper with a number 40 blade.
		<ol>
			<li>In case of depilatory agent, apply this compound to the surgical area for 2-3 min and then remove it with cotton. We use a commercially available compound of calcium hydroxide and sodium hydroxide.</li>
		</ol>
		</li>
		<li>Prep the area over the shoulder with 70% alcohol and subcutaneously inject carprofen (5 mg/kg) with a 25 gauge or smaller bore needle. This treatment will provide postoperative analgesia for the animal.</li>
		<li>Transfer the animal to the surgical field and position in dorsal recumbency.</li>
		<li>Prepare the surgical site by scrubbing 8-12% providone-iodine with a clean cotton applicator or cotton gauze. Then rinse the skin twice with 70% alcohol.</li>
		<li>Place ocular lubricant in both eyes to reduce the incidence of corneal desiccation. Cover the surgical site with a sterile drape.</li>
	</ol>
	</li>
	<li>Operative Technique
	<ol>
		<li>Make a median abdominal laparotomy incision approximately 2-2.5 cm in length with a scalpel beginning immediately caudal the xiphoid process and extending towards the pelvis.</li>
		<li>Mobilize the small bowel and duodenum and reflect laterally to the right. Roll up a strip of sterile cotton gauzed and soaked with sterile saline for injection to allow packing of the viscera to improve exposure.</li>
		<li>With the small intestine mobilized to the right side of the abdomen, expose the retroperitoneum and expose the abdominal aorta from the left renal vein to the aortic bifurcation (Figure 1).</li>
		<li>With a sterile cotton-tipped applicator, deliver 30 consecutive crushes, each five seconds in duration.</li>
		<li>Remove the packing and allow the viscera to return to their native position.</li>
		<li>Close fascia with a running 4-0 absorbable monofilament suture (polydioxanone). Skin is closed with a running 6-0 non-absorbable monofilament suture (nylon).</li>
	</ol>
	</li>
	<li>Recovery and Post-Procedure Care
	<ol>
		<li>After the procedure, place the animal in a recovery cage with clean bedding on an isothermal pad to continue thermal support until the animal is able to ambulate normally. Do not leave the animal unattended until it demonstrates the ability to maintain sternal recumbency.</li>
		<li>Monitor the animal every hour for the first 4 h after surgery. Once the animal is ambulating normally return it to the assigned husbandry room. The animal will not be housed in the company of another animal until it has fully recovered.</li>
		<li>Monitor the animal twice daily for the first 72 h after surgery and at least 3 times a week thereafter. Monitoring includes weighing the animal three times a week.</li>
		<li>Administer carpofen (5 mg/kg) subcutaneously twice daily for the first 72 h after surgery.</li>
	</ol>
	</li>
</ol>
2. Procurement of Tissue
<ol>
	<li>Method of Euthanasia
	<ol>
		<li>Euthanize the animals at predetermined time points.</li>
		<li>Induce anesthesia via an induction tank with 100% O2 and 2.5% isoflurane delivered via precision vaporizer.
		<ol>
			<li>To study the integrity of the endothelium, Administer Evans Blue dye to the animal. 
			NOTE: Evans Blue is a negatively charged azo dye with a high binding affinity for albumin and can only stain blood vessels in the absence of an intact endothelium6.</li>
			<li>For endothelial integrity studies, at the time of euthanasia, perfuse the animals with 5 mL of 0.3 % Evans Blue dye followed by 5 mL PBS at physiological pressure for 5 min. The animal is maintained in a surgical plane of anesthesia during the perfusion procedure.</li>
		</ol>
		</li>
		<li>After achieving a deep anesthetic plane, open the chest with a sternotomy. Make a laceration in the right atrium to allow drainage of blood from the animal and access the left ventricle is accessed with a 21 G needle and inject phosphate buffered saline (PBS) until the effluent from the right atrium is clear.</li>
		<li>After perfusion with PBS, enter the abdomen through a midline incision.</li>
		<li>Once again, mobilize the small intestine to the right side of the abdomen exposing the infrarenal aorta, sharply dissect the aorta from adjacent tissues and excise it from the left renal vein to the aortic bifurcation.</li>
		<li>Store the excised aorta in a 4% paraformaldehyde solution until tissue processing occurs.
		<ol>
			<li>For endothelial integrity studies, open the aorta longitudinally and pin it to a wax sheet exposing the entirety of the luminal surface6. Carry out a qualitative assessment of endothelial integrity by the degree of staining with Evans Blue dye6.</li>
			<li>For other histologic assays, section the aorta transversely and embed it in optimal cutting temperature (OCT) compounds as dictated by the histological method to be used 7.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Carmeliet, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+angiogenesis+and+arteriogenesis.">Mechanisms of angiogenesis and arteriogenesis.</a> Nat Med. 6 (4), 389-395 (2000).</li><li>Carmeliet, P., Moons, L., Collen, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+models+of+angiogenesis,+arterial+stenosis,+atherosclerosis+and+hemostasis.">Mouse models of angiogenesis, arterial stenosis, atherosclerosis and hemostasis.</a> Cardiovasc Res. 39 (1), 8-33 (1998).</li><li>Baker, A. 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=Heparanase+Alters+Arterial+Structure,+Mechanics,+and+Repair+Following+Endovascular+Stenting+in+Mice.">Heparanase Alters Arterial Structure, Mechanics, and Repair Following Endovascular Stenting in Mice.</a> Circ Res. 104 (3), 380-387 (2009).</li><li>Petrov, L., Laurila, H., Hayry, P., Vamvakopoulos, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+mouse+model+of+aortic+angioplasty+for+genomic+studies+of+neointimal+hyperplasia.">A mouse model of aortic angioplasty for genomic studies of neointimal hyperplasia.</a> J Vasc Res. 42 (4), 292-300 (2005).</li><li>Li, 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=Vascular+smooth+muscle+cells+of+recipient+origin+mediate+intimal+expansion+after+aortic+allotransplantation+in+mice.">Vascular smooth muscle cells of recipient origin mediate intimal expansion after aortic allotransplantation in mice.</a> Am J Path. 158 (6), 1943-1947 (2001).</li><li>Radu, M., Chernoff, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+in+vivo+assay+to+test+blood+vessel+permeability.">An in vivo assay to test blood vessel permeability.</a> J Vis Exp. (73), e50062 (2013).</li><li>Turbett, G. R., Sellner, L. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+optimal+cutting+temperature+compound+can+inhibit+amplification+by+polymerase+chain+reaction.">The use of optimal cutting temperature compound can inhibit amplification by polymerase chain reaction.</a> Diagn Mol Pathol. 6 (5), 298-303 (1997).</li><li>Puchtler, H., Waldrop, F. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+mechanism+of+Verhoeff's+elastica+stain:+a+convenient+stain+for+myelin+sheaths.">On the mechanism of Verhoeff's elastica stain: a convenient stain for myelin sheaths.</a> Histochem. 62 (3), 233-247 (1979).</li><li>Salic, A., Mitchison, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+chemical+method+for+fast+and+sensitive+detection+of+DNA+synthesis+in+vivo.">A chemical method for fast and sensitive detection of DNA synthesis in vivo.</a> Proc Natl Acad Sci U S A. 105 (7), 2415-2420 (2008).</li><li>Nelson, P. R., Yamamura, S., Mureebe, L., Itoh, H., 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=Smooth+muscle+cell+migration+and+proliferation+are+mediated+by+distinct+phases+of+activation+of+the+intracellular+messenger+mitogen-activated+protein+kinase.">Smooth muscle cell migration and proliferation are mediated by distinct phases of activation of the intracellular messenger mitogen-activated protein kinase.</a> J Vasc Surg. 27 (1), 117-125 (1998).</li><li>Rzucidlo, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signaling+pathways+regulating+vascular+smooth+muscle+cell+differentiation.">Signaling pathways regulating vascular smooth muscle cell differentiation.</a> Vascular. 17, S15-S20 (2009).</li><li>Aoki, T., Sumii, T., Mori, T., Wang, X., Lo, E. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood-brain+barrier+disruption+and+matrix+metalloproteinase-9+expression+during+reperfusion+injury:+mechanical+versus+embolic+focal+ischemia+in+spontaneously+hypertensive+rats.">Blood-brain barrier disruption and matrix metalloproteinase-9 expression during reperfusion injury: mechanical versus embolic focal ischemia in spontaneously hypertensive rats.</a> Stroke. 33 (11), 2711-2717 (2002).</li><li>Yu, 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=MARCKS+Signaling+Differentially+Regulates+Vascular+Smooth+Muscle+and+Endothelial+Cell+Proliferation+through+a+KIS-,+p27kip1-+Dependent+Mechanism.">MARCKS Signaling Differentially Regulates Vascular Smooth Muscle and Endothelial Cell Proliferation through a KIS-, p27kip1- Dependent Mechanism.</a> PLoS One. 10 (11), e0141397 (2015).</li><li>Banai, 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=Rabbit+ear+model+of+injury-induced+arterial+smooth-muscle+cell-proliferation+-+kinetics,+reproducibility,+and+implications.">Rabbit ear model of injury-induced arterial smooth-muscle cell-proliferation - kinetics, reproducibility, and implications.</a> Circ Res. 69 (3), 748-756 (1991).</li></ol>

This work was funded by the Department of Veterans Affairs Career Development Award (1IK2BX001553-01) (TSM) and the Vascular Cures E. J. Wylie Scholarship (TSM).

We have characterized the effects of a murine aortic injury model that results in medial hyperplasia and endothelial barrier dysfunction. Partial EC detachment along the aorta intima accompanied the loss of cell-cell contact and enhancement of cell protrusions. Correspondingly, endothelial barrier function was significantly impaired, which stimulated the mitogen-sensitive signaling pathways, leading to proliferation of VSMCs and thickening of the vessel wall. The strengths of this model is that it is technically easier t...

Restenosis following cardiovascular procedures (bypass surgery, angioplasty, or stenting) is a significant problem reducing the durability of these procedures. All revascularization procedures are plagued by restenosis. Present strategies to prevent restenosis (drug-eluting stents and drug-coated balloons) inhibit both vascular smooth muscle cell (VSMC) and endothelial cell proliferation (EC). Consequently, these interventions prevent VSMC mediated restenosis, but also prevent the regeneration of the endothelium. Without an intact endothelium, patients are required to be on potent antiplatelet agents to decrease the risk of in situ thrombosis at the risk of bleeding complications. An ideal therapy would inhibit VSMC proliferation while promoting regeneration of the endothelium. Thus, there is a need to simultaneously study VSMC proliferation and endothelial barrier function in vivo.
Presently, there are several mouse models of restenosis 1. These models include carotid ligation and femoral artery wire injury 2. Aortic models include stent placement 3, balloon injury 4, and aortic allograft 5. All of the present models are limited. Carotid ligation generates a flow-mediated neointimal lesion and does not have endothelial injury. Additionally, both carotid and femoral arteries have many fold fewer cell layers than human vessels, limiting their translational value. The mouse aorta which is approximately 1.3 mm in diameter, is the only vessel that approximates a clinically relevant (coronary) human artery (3).
Despite the translational potential of murine aortic models of disease, current models have limitations. These models require advanced microsurgical skills and specialized equipment such as angioplasty balloons and stents. Herein, we present a novel, reproducible technique to simultaneously induce VSMC proliferation and disrupt endothelial barrier function.

<table><tbody><tr><td>Ocular lubricant</td><td>Dechra</td><td>17033-211-38</td><td>Pharmaceutical agents</td></tr><tr><td>Isoflurane</td><td>VetOne</td><td>502017</td><td>Pharmaceutical agents</td></tr><tr><td>Carprofen</td><td>Zoetis</td><td>26357</td><td>Pharmaceutical agents</td></tr><tr><td>Precision vaporizer</td><td>Summit Medical</td><td>10675</td><td>Surgical supplies</td></tr><tr><td>Charcoal scavenger</td><td>Bickford Inc.</td><td>80120</td><td>Surgical supplies</td></tr><tr><td>Isothermal pad</td><td>Harvard Apparatus</td><td>50-7053-R</td><td>Surgical supplies</td></tr><tr><td>Sterile cotton-tipped applicator</td><td>Fisher Scientific</td><td>23-400-124</td><td>Surgical supplies</td></tr><tr><td>4-0 absorbable monofilament suture&amp;nbsp;</td><td>Ethicon, Inc</td><td>J310</td><td>Surgical supplies</td></tr><tr><td>5-0 non-absorbable monofilament suture</td><td>Ethicon,Inc</td><td>1666</td><td>Surgical supplies</td></tr><tr><td>21-gauge x 1 inch needle</td><td>BD Biosciences</td><td>305165</td><td>Surgical supplies</td></tr><tr><td>25-gauge x 1 inch&amp;nbsp; needle</td><td>BD Biosciences</td><td>305125</td><td>Surgical supplies</td></tr><tr><td>Dry sterilizer</td><td>Cellpoint&amp;nbsp;</td><td>7770</td><td>Surgical supplies</td></tr><tr><td>Fine scissors</td><td>Fine Science Tools</td><td>14058-09</td><td>Surgical instruments</td></tr><tr><td>Adson forceps</td><td>Fine Science Tools</td><td>11006-12</td><td>Surgical instruments</td></tr><tr><td>Dumont #5 fine forceps</td><td>Fine Science Tools</td><td>11254-20</td><td>Surgical instruments</td></tr><tr><td>Vannas Spring Scissors 3 mm cutting edge</td><td>Fine Science Tools</td><td>15000-00</td><td>Surgical instruments</td></tr><tr><td>Needle driver</td><td>Fine Science Tools</td><td>91201-13</td><td>Surgical instruments</td></tr><tr><td>Scalpel handle #4</td><td>Fine Science Tools</td><td>10004-13</td><td>Surgical instruments</td></tr><tr><td>Scalpel blades #10</td><td>Fine Science Tools</td><td>10010-00</td><td>Surgical instruments</td></tr><tr><td>PBS&amp;nbsp;</td><td>Lonza</td><td>17-516F</td><td>Reagents for tissue processing</td></tr><tr><td>Evans Blue</td><td>Sigma-Aldrich</td><td>E2129</td><td>Reagents for tissue processing</td></tr><tr><td>Paraformaldehyde</td><td>Sigma-Aldrich</td><td>P6148</td><td>Reagents for tissue processing</td></tr><tr><td>Modeling wax</td><td>Bego</td><td>40001</td><td>Reagents for tissue processing</td></tr><tr><td>OCT compound</td><td>Tissue-Tek Sakura</td><td>4583</td><td>Reagents for tissue processing</td></tr><tr><td>Mayer&#039;s hematoxylin solution</td><td>Sigma-Aldrich</td><td>MHS16</td><td>Reagents for immunohistological analysis</td></tr><tr><td>Eosin Y solution alcoholic&amp;nbsp;</td><td>Sigma-Aldrich</td><td>HT110316</td><td>Reagents for immunohistological analysis</td></tr><tr><td>Elastin stain kit</td><td>Sigma-Aldrich</td><td>HT25A</td><td>Reagents for immunohistological analysis</td></tr><tr><td>Click-it Edu Alexa-488 Imaging Kit</td><td>Invitrogen</td><td>C10337</td><td>Reagents for immunohistological analysis</td></tr><tr><td>Anti-Erk1/2 antibody</td><td>Cell Signaling Technology</td><td>4695</td><td>Reagents for immunohistological analysis</td></tr><tr><td>Anti-phospho-Erk1/2 antibody</td><td>Cell Signaling Technology</td><td>4370</td><td>Reagents for immunohistological analysis</td></tr><tr><td>Anti-p27kip1 antibody</td><td>Cell Signaling Technology</td><td>3698</td><td>Reagents for immunohistological analysis</td></tr><tr><td>Trichloroacetic acid</td><td>Sigma-Aldrich</td><td>T9159</td><td>Reagents for immunohistological analysis</td></tr></tbody></table>

murine aortic crush injury: an efficient in vivo model of smooth muscle cell proliferation and endothelial function

Arterial reconstruction, whether angioplasty or bypass surgery, involves iatrogenic trauma causing endothelial disruption and vascular smooth muscle cell (VSMC) proliferation. Common murine models study small vessels such as the carotid and femoral arteries. Herein we describe an in vivo system in which both VSMC proliferation and endothelial barrier function can be simultaneously assessed in a large vessel. We studied the infrarenal aortic response to injury in C57BL/6 mice. The aorta was injured from the left renal vein to the aortic bifurcation by 30 transmural crushes of 5-seconds duration with a cotton-tipped applicator. Morphological changes were assessed with conventional histology. Aorta wall thickness was measured from the luminal surface to the adventitia. EdU integration and counter staining with DAPI and alpha-actin was used to demonstrate VSMC proliferation. Activation of ERK1/2, a known moderator of intimal hyperplasia formation, was determined by Western Blot analysis. The effect of inflammation was determined by immunohistochemistry for B-cells, T-cells, and macrophages. En face sections of endothelium were visualized with scanning electron microscopy (SEM). Endothelial barrier function was determined with Evans Blue staining. Transmural injury resulted in aortic wall thickening. This injury induced VSMC proliferation, most prominently at 3 days after injury, and early activation of ERK1/2 and decreased p27kip1 expression. Injury did not result in increased B-cells, T-cells, or macrophages infiltration in the vessel wall. Injury caused partial endothelial cell denudation and loss of cell-cell contact. Injury resulted in a significant loss of endothelial barrier function, which returned to baseline after seven days. The murine transmural blunt aortic injury model provides an efficient system to simultaneously study both VSMC proliferation and endothelial barrier function in a large vessel.

Transverse sections aorta embedded in OCT were sectioned, and stained with hematoxylin and eosin then counter stained with Verhoeff-Van Gieson (VVG) stain to identify the internal and external elastic lamina 7. Crush injury induced aortic wall thickening compared to the aortas of animals treated with a sham procedure (laparotomy and small bowel mobilization alone). Wall thickness, as assessed by the distance from adventitia to the lumen, was greatest three days aft...

Watch this Scientific Journal Video about Murine Aortic Crush Injury: An Efficient In Vivo Model of Smooth Muscle Cell Proliferation and Endothelial Function at JoVE.com

Murine Aortic Crush Injury: An Efficient In Vivo Model of Smooth Muscle Cell Proliferation and Endothelial Function

Restenosis following cardiovascular procedures (bypass surgery, angioplasty, or stenting) is a significant problem reducing the durability of these procedures. An ideal therapy would inhibit smooth muscle cell (VSMC) proliferation while promoting regeneration of the endothelium. We describe a model for simultaneous assessment of VSMC proliferation and endothelial function in vivo.

Murine Aortic Crush Injury: An Efficient In Vivo Model of Smooth Muscle Cell Proliferation and Endothelial Function

Arterial reconstruction, whether angioplasty or bypass surgery, involves iatrogenic trauma causing endothelial disruption and vascular smooth muscle cell ...

murine-aortic-crush-injury-an-efficient-vivo-model-smooth-muscle-cell

Research

JoVE Journal

Biology

8.3K Views.  Baltimore Veterans Affairs Medical Center.  Restenosis na cardiovasculaire procedures (bypass operatie, angioplastie of stenting) is een significant probleem waardoor de duurzaamheid van deze procedures wordt verminderd. Een ideale therapie zou de proliferatie van gladde spiercellen (VSMC) verhinderen, terwijl het bevorderen van regeneratie van het endothelium. We beschrijven een model voor gelijktijdige beoordeling van VSMC proliferatie en endotheliale functie in vivo. 

Video: Murine Aortic Crush Injury: An Efficient In Vivo Model of Smooth Muscle Cell Proliferation and Endothelial Function

Using In Vivo and Tissue and Cell Explant Approaches to Study the Morphogenesis and Pathogenesis of the Embryonic and Perinatal Aorta

The aorta is the largest artery in the body. The aortic wall is composed of an inner layer of endothelial cells, a middle layer of alternating elastic lamellae and smooth muscle cells (SMCs), and an outer layer of fibroblasts and extracellular matrix. In contrast to the widespread study of pathological models (e.g., atherosclerosis) in the adult aorta, much less is known about the embryonic and perinatal aorta. Here, we focus on SMCs and provide protocols for the analysis of the morphogenesis and pathogenesis of embryonic and perinatal aortic SMCs in normal development and disease. Specifically, the four protocols included are: i) in vivo embryonic fate mapping and clonal analysis; ii) explant embryonic aorta culture; iii) SMC isolation from the perinatal aorta; and iv) subcutaneous osmotic mini-pump placement in pregnant (or non-pregnant) mice. Thus, these approaches facilitate the investigation of the origin(s), fate, and clonal architecture of SMCs in the aorta in vivo. They allow for modulating embryonic aorta morphogenesis in utero by continuous exposure to pharmacological agents. In addition, isolated aortic tissue explants or aortic SMCs can be used to gain insights into the role of specific gene targets during fundamental processes such as muscularization, proliferation, and migration. These hypothesis-generating experiments on isolated SMCs and the explanted aorta can then be assessed in the in vivo context through pharmacological and genetic approaches.

Using In Vivo and Tissue and Cell Explant Approaches to Study the Morphogenesis and Pathogenesis of the Embryonic and Perinatal Aorta

Protocols for studying the embryonic and perinatal murine aorta using in vivo clonal analysis and fate mapping, aortic explants, and isolated smooth muscle cells are detailed here. These diverse approaches facilitate the investigation of the morphogenesis of the embryonic and perinatal aorta in normal development and the pathogenesis in disease.

The aorta is the largest artery in the body. The aortic wall is composed of an inner layer of endothelial cells, a middle layer of alternating elastic ...

Developmental Biology

Murine Spinotrapezius Model to Assess the Impact of Arteriolar Ligation on Microvascular Function and Remodeling

The murine spinotrapezius is a thin, superficial skeletal support muscle that extends from T3 to L4, and is easily accessible via dorsal skin incision. Its unique anatomy makes the spinotrapezius useful for investigation of ischemic injury and subsequent microvascular remodeling. Here, we demonstrate an arteriolar ligation model in the murine spinotrapezius muscle that was developed by our research team and previously published1-3. For certain vulnerable mouse strains, such as the Balb/c mouse, this ligation surgery reliably creates skeletal muscle ischemia and serves as a platform for investigating therapies that stimulate revascularization. Methods of assessment are also demonstrated, including the use of intravital and confocal microscopy. The spinotrapezius is well suited to such imaging studies due to its accessibility (superficial dorsal anatomy) and relative thinness (60-200 &mu;m). The spinotrapezius muscle can be mounted en face, facilitating imaging of whole-muscle microvascular networks without histological sectioning. We describe the use of intravital microscopy to acquire metrics following a functional vasodilation procedure; specifically, the increase in arterilar diameter as a result of muscle contraction. We also demonstrate the procedures for harvesting and fixing the tissues, a necessary precursor to immunostaining studies and the use of confocal microscopy.

We demonstrate a novel arterial ligation model in murine spinotrapezius muscle, including a step-by-step procedure and description of required instrumentation. We describe the surgery and relevant outcome measurements relating to vascular network remodeling and functional vasodilation using intravital and confocal microscopy.

The murine spinotrapezius is a thin, superficial skeletal support muscle that extends from T3 to L4, and is easily accessible via dorsal skin incision. ...

Medicine

A New Murine Model of Endovascular Aortic Aneurysm Repair

Endovascular aneurysm exclusion is a validated technique to prevent aneurysm rupture. Long-term results highlight technique limitations and new aspects of Abdominal aortic aneurysm (AAA) pathophysiology. There is no abdominal aortic aneurysm endograft exclusion model cheap and reproducible, which would allow deep investigations of AAA before and after treatment. We hereby describe how to induce, and then to exclude with a covered coronary stentgraft an abdominal aortic aneurysm in a rat. The well known elastase induced AAA model was first reported in 19901 in a rat, then described in mice2. Elastin degradation leads to dilation of the aorta with inflammatory infiltration of the abdominal wall and intra luminal thrombus, matching with human AAA. Endovascular exclusion with small covered stentgraft is then performed, excluding any interactions between circulating blood and the aneurysm thrombus. Appropriate exclusion and stentgraft patency is confirmed before euthanasia by an angiography thought the left carotid artery. Partial control of elastase diffusion makes aneurysm shape different for each animal. It is difficult to create an aneurysm, which will allow an appropriate length of aorta below the aneurysm for an easy stentgraft introduction, and with adequate proximal and distal neck to prevent endoleaks. Lots of failure can result to stentgraft introduction which sometimes lead to aorta tear with pain and troubles to stitch it, and endothelial damage with post op aorta thrombosis. Giving aspirin to rats before stentgraft implantation decreases failure rate without major hemorrhage. Clamping time activates neutrophils, endothelium and platelets, and may interfere with biological analysis.

Histological and biochemical modifications after aneurysm endograft exclusion are still unclear. We describe a new model of endograft implantation on a murine aneurysm. Through thrombus analysis with and without persistant circulating blood stream interface, and new endovascular biomaterials evaluation, this model will help to better understand endovascular aneurysm exclusion pathobiology.

Endovascular aneurysm exclusion is a validated technique to prevent aneurysm rupture. Long-term results highlight technique limitations and new aspects of ...

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

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

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

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

Balloon-based Injury to Induce Myointimal Hyperplasia in the Mouse Abdominal Aorta

The use of animal models is essential for a better understanding of MH, one major cause for arterial stenosis.In this article, we demonstrate a murine balloon denudation model, which is comparable with established vessel injury models in large animals. The aorta denudation model with balloon catheters mimics the clinical setting and leads to comparable pathobiological and physiological changes. Briefly, after performing a horizontal incision in the aorta abdominalis, a balloon catheter will be inserted into the vessel, inflated, and introduced retrogradely. Inflation of the balloon will lead to intima injury and overdistension of the vessel. After removing the catheter, the aortic incision will be closed with single stiches. The model shown in this article is reproducible, easy to perform, and can be established quickly and reliably. It is especially suitable for evaluating expensive experimental therapeutic agents, which can be applied in an economical fashion. By using different knockout-mouse strains, the impact of different genes on MH development can be assessed.

This article demonstrates a murine model to study the development of myointimal hyperplasia (MH) after aortic balloon injury.

The use of animal models is essential for a better understanding of MH, one major cause for arterial stenosis.In this article, we demonstrate a murine ...

Construction of a Human Aorta Smooth Muscle Cell Organ-On-A-Chip Model for Recapitulating Biomechanical Strain in the Aortic Wall

Conventional two-dimensional cell culture techniques and animal models have been used in the study of human thoracic aortic aneurysm and dissection (TAAD). However, human TAAD sometimes cannot be characterized by animal models. There is an apparent species gap between clinical human studies and animal experiments that may hinder the discovery of therapeutic drugs. In contrast, the conventional cell culture model is unable to simulate in vivo biomechanical stimuli. To this end, microfabrication and microfluidic techniques have developed greatly in recent years, providing novel techniques for establishing organoids-on-a-chip models that replicate the biomechanical microenvironment. In this study, a human aorta smooth muscle cell organ-on-a-chip (HASMC-OOC) model was developed to simulate the pathophysiological parameters of aortic biomechanics, including the amplitude and frequency of cyclic strain experienced by human aortic smooth muscle cells (HASMCs) that play a vital role in TAAD. In this model, the morphology of HASMCs became elongated in shape, aligned perpendicularly to the strain direction, and presented a more contractile phenotype under strain conditions than under static conventional conditions. This was consistent with the cell orientation and phenotype in native human aortic walls. Additionally, using bicuspid aortic valve-related TAAD (BAV-TAAD) and tricuspid aortic valve-related TAAD (TAV-TAAD) patient-derived primary HASMCs, we established BAV-TAAD and TAV-TAAD disease models, which replicate HASMC characteristics in TAAD. The HASMC-OOC model provides a novel in vitro platform that is complementary to animal models for further exploring the pathogenesis of TAAD and discovering therapeutic targets.

Here, we developed a human aorta smooth muscle cell organ-on-a-chip model to replicate the in vivo biomechanical strain of smooth muscle cells in the human aortic wall.

Conventional two-dimensional cell culture techniques and animal models have been used in the study of human thoracic aortic aneurysm and dissection ...

Bioengineering

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

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

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

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

Quantitative Analysis of Cellular Composition in Advanced Atherosclerotic Lesions of Smooth Muscle Cell Lineage-Tracing Mice

Atherosclerosis remains the leading cause of death worldwide and, despite countless preclinical studies describing promising therapeutic targets, novel interventions have remained elusive. This is likely due, in part, to a reliance on preclinical prevention models investigating the effects of genetic manipulations or pharmacological treatments on atherosclerosis development rather than the established disease. Also, results of these studies are often confounding because of the use of superficial lesion analyses and a lack of characterization of lesion cell populations. To help overcome these translational hurdles, we propose an increased reliance on intervention models that employ investigation of changes in cellular composition at a single cell level by immunofluorescent staining and confocal microscopy. To this end, we describe a protocol for testing a putative therapeutic agent in a murine intervention model including a systematic approach for animal dissection, embedding, sectioning, staining, and quantification of brachiocephalic artery lesions. In addition, due to the phenotypic diversity of cells within late-stage atherosclerotic lesions, we describe the importance of using cell-specific, inducible lineage tracing mouse systems and how this can be leveraged for unbiased characterization of atherosclerotic lesion cell populations. Together, these strategies may assist vascular biologists to more accurately model therapeutic interventions and analyze atherosclerotic disease and will hopefully translate into a higher rate of success in clinical trials.

We propose a standardized protocol to characterize the cellular composition of late-stage murine atherosclerotic lesions including systematic methods of animal dissection, tissue embedding, sectioning, staining, and analysis of brachiocephalic arteries from atheroprone smooth muscle cell lineage tracing mice.

Atherosclerosis remains the leading cause of death worldwide and, despite countless preclinical studies describing promising therapeutic targets, novel ...

Murine Cervical Aortic Transplantation Model using a Modified Non-Suture Cuff Technique

With the introduction of powerful immunosuppressive protocols, distinct advances are possible in the prevention and therapy of acute rejection episodes. However, only minor improvement in the long-term results of transplanted solid organs could be observed over the past decades. In this context, chronic allograft vasculopathy (CAV) still represents the leading cause of late organ failure in cardiac, renal and pulmonary transplantation.
Thus far, the underlying pathogenesis of CAV development remains unclear, explaining why effective treatment strategies are presently missing and emphasizing a need for relevant experimental models in order to study the underlying pathophysiology leading to CAV formation. The following protocol describes a murine heterotopic cervical aortic transplantation model using a modified non-suture cuff technique. In this technique, a segment of the thoracic aorta is interpositioned in the right common carotid artery. With the use of the non-suture cuff technique, an easy to learn and reproducible model can be established, minimizing the possible heterogeneity of sutured vascular micro anastomoses.

Here, we present a protocol of heterotopic aortic transplantation in mice using the non-suture cuff technique in a cervical murine model. This model can be used to study the underlying pathology of chronic allograft vasculopathy (CAV) and can help evaluate new therapeutic agents in order to prevent its formation.

With the introduction of powerful immunosuppressive protocols, distinct advances are possible in the prevention and therapy of acute rejection episodes. ...

Isolation of Primary Patient-specific Aortic Smooth Muscle Cells and Semiquantitative Real-time Contraction Measurements In Vitro

Smooth muscle cells (SMCs) are the predominant cell type in the aortic media. Their contractile machinery is important for the transmission of force in the aorta and regulates vasoconstriction and vasodilation. Mutations in genes encoding for the SMC contractile apparatus proteins are associated with aortic diseases, such as thoracic aortic aneurysms. Measuring SMC contraction in vitro is challenging, especially in a high-throughput manner, which is essential for screening patient material. Currently available methods are not suitable for this purpose. This paper presents a novel method based on electric cell-substrate impedance sensing (ECIS). First, an explant protocol is described to isolate patient-specific human primary SMCs from aortic biopsies and patient-specific human primary dermal fibroblasts for the study of aortic aneurysms. Next, a detailed description of a new contraction method is given to measure the contractile response of these cells, including the subsequent analysis and suggestion for comparing different groups. This method can be used to study the contraction of adherent cells in the context of translational (cardiovascular) studies and patient and drug screening studies.

Isolation of Primary Patient-specific Aortic Smooth Muscle Cells and Semiquantitative Real-time Contraction Measurements In Vitro

This paper describes an explant culture-based method for the isolation and culturing of primary, patient-specific human aortic smooth muscle cells and dermal fibroblasts. Furthermore, a novel method is presented for measuring cell contraction and subsequent analysis, which can be used to study patient-specific differences in these cells.

Smooth muscle cells (SMCs) are the predominant cell type in the aortic media. Their contractile machinery is important for the transmission of force in ...

Murine Aortic Crush Injury: An Efficient In Vivo Model of Smooth Muscle Cell Proliferation and Endothelial Function

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