Name: reproducible arterial denudation injury by infrarenal abdominal aortic clamping in a murine model
Uploaded: 2016-11-24T12:30:00
Description: Watch this Scientific Journal Video about Reproducible Arterial Denudation Injury by Infrarenal Abdominal Aortic Clamping in a Murine Model at JoVE.com

This work was supported by grants from the Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research at UCLA Training Program to ASS and AIM, Philip J. Whitcome Fellowship to AIM, and National Institutes of Health (HL130290) to MLIA.

NOTE: This protocol has been approved by the Animal Research Committee at the University of California Los Angeles.
1. Preoperative Preparation and Anesthesia
<ol>
	<li>Be sure to observe sterile technique throughout the procedure.</li>
	<li>Sterilize all surgical supplies using a steam autoclave.</li>
	<li>Turn on heated rodent surgical platform prior to anesthetic induction so that it may warm to appropriate temperature (37 &#176;C) and place under stereomicroscope for visualization during surgical procedure.<...

<ol>
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Arterial denudation injury due to percutaneous interventions, such as balloon angioplasty and vascular stenting, result in early and late vascular thrombosis and restenosis and contributes to recurrent ischemic events3,12. Interestingly, surgical vascular clamping has also been implicated as a cause of arterial denudation, expanding the scope of the problem to patients undergoing any vascular procedure, whether percutaneous or open13. While impaired re-endothelialization is a fairly recognized cause...

Thrombosis and restenosis are serious early and late complications in patients who undergo percutaneous vascular interventions, such as endovascular balloon angioplasty and stenting1,2. Several strategies have been employed to address these complications, particularly dual antiplatelet therapy and drug-eluting stents. However, little focus has been placed on the underlying cause of thrombosis and restenosis, namely the loss of endothelial cell coverage (denudation). Denudation injury is an inevitable consequence of interventional procedures due to mechanical trauma to the blood vessel wall. This mechanical trauma can result in damage and removal of the protective endothelial layer and exposure of the basement membrane and vascular smooth muscle to circulating blood3. The loss of endothelial cells in these areas creates a pro-thrombotic and pro-inflammatory environment that not only promotes platelet adhesion and subsequent thrombosis, but also stimulates migration and proliferation of vascular smooth muscle cells resulting in neointimal thickening and restenosis4. These complications, and their associated therapies, lead to significant morbidity, most notably recurrent ischemic disease and bleeding events that impact human health.
Re-endothelialization of the denuded injury from the wound margins is of paramount importance in preventing thrombosis and restenosis5. Autopsy results and animal models have effectively demonstrated reduced rates of thrombosis with coverage of stent struts6,7. Drug-eluting stents, devised to decrease rates of restenosis by inhibiting smooth muscle proliferation and neointimal hyperplasia, result in significant impairments in arterial re-endothelialization and increasing rates of late thrombosis3. Unfortunately, understanding the mechanisms for re-endothelialization has been a slow process, largely limited by the lack of appropriate animal models8.
Several animal models for understanding the roles of endothelial cells and vascular smooth muscle cells following arterial injury have been created7,9,10. The rat carotid artery balloon injury model is the best characterized and has been employed to study the effects of denudation injury at the gross, cellular and molecular level11. Nevertheless, a highly reproducible murine model of arterial denudation injury with excellent survival rates is missing and much needed to take advantage of&#160;multiple transgenic lines available to better elucidate vascular regeneration in multiple settings.
This manuscript presents a murine model of arterial denudation injury that is reproducible and simple to perform. The approach has shown minimal morbidity and mortality in several transgenic lines. Because of the broad number of genetically modified mouse lines, this model may be used to elucidate the molecular mechanisms underlying re-endothelialization after denudation injury.

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			<td height="40" style="height:40px;width:289px;">Zeiss Discovery.V12 Stereomicroscope</td>
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			<td height="20" style="height:20px;width:289px;">Rodent Heated Surgical Platform</td>
			<td style="width:187px;">Protech International</td>
			<td style="width:119px;">RES4000</td>
			<td>Heated platform for body temperature maintenance with nosecone for anesthestic maintenance and on which surgical procedure is performed</td>
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			<td height="20" style="height:20px;">Oster&#8482; A5 Professional Animal Clipper</td>
			<td>M.Schneider &#38; Sons Inc.</td>
			<td>78005010</td>
			<td>Use with animal clipper size 40</td>
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			<td height="20" style="height:20px;width:289px;">Adjustable Wire Retractor</td>
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			<td>Fine Science Tools</td>
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			<td height="20" style="height:20px;width:289px;">0.5% Marcaine</td>
			<td style="width:187px;">Hospira</td>
			<td style="width:119px;">0409-1610-50</td>
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			<td height="20" style="height:20px;width:289px;">5-0 Suture, Vicryl</td>
			<td style="width:187px;">Fisher Scientific</td>
			<td style="width:119px;">NC0189890</td>
			<td>tapered needle</td>
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			<td height="20" style="height:20px;width:289px;">Vetbond</td>
			<td style="width:187px;">Fisher Scientific</td>
			<td style="width:119px;">NC0304169</td>
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			<td height="40" style="height:40px;width:289px;">Falcon&#174; 35 mm Not TC-Treated Easy-Grip Style Bacteriological Petri Dish</td>
			<td style="width:187px;">Corning</td>
			<td align="right">351008</td>
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			<td height="20" style="height:20px;width:289px;">Sylgard 184 Silicone Elastomer Kit</td>
			<td style="width:187px;">Dow Corning</td>
			<td style="width:119px;">3097358-1004</td>
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			<td height="20" style="height:20px;width:289px;">Dissecting pins</td>
			<td style="width:187px;">Fisher Scientific</td>
			<td>NC9681411</td>
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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.

Eighty-five mice have undergone the survival surgical technique described in this report for infrarenal abdominal aortic clamping. The overall survival rate was 85.9%. Operative complications included intestinal bleeding and great vessel perforation, resulting in 5.9% and 3.5% mortality respectively (Table 1). After recovery from anesthesia, mice walk normally and show no signs of ischemic injury to the inferior limbs. No weight loss or lack of appetite were noted.
<p...

Watch this Scientific Journal Video about Reproducible Arterial Denudation Injury by Infrarenal Abdominal Aortic Clamping in a Murine Model at JoVE.com

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

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 ...

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