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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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+Model+of+Arterial+Injury.">Mouse Model of Arterial Injury.</a> Circ Res. 73, 792-796 (1993).</li><li>Kumar, A., Lindner, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Remodeling+with+neointima+formation+in+the+mouse+carotid+artery+after+cessation+of+blood+flow.">Remodeling with neointima formation in the mouse carotid artery after cessation of blood flow.</a> Arterioscler Thromb Vasc Biol. 17, 2238-2244 (1997).</li><li>Hehrlein, C., Weinschenk, I., Metz, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long+period+of+balloon+inflation+and+the+implantation+of+stents+potentiate+smooth+muscle+cell+death.+Possible+role+of+chronic+vascular+injury+in+restenosis.">Long period of balloon inflation and the implantation of stents potentiate smooth muscle cell death. 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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>
			<td style="width:187px;">Zeiss</td>
			<td colspan="2" style="width:285px;">495037-9904-000</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;width:289px;">Isoflurane</td>
			<td style="width:187px;">Henry Schein</td>
			<td style="width:119px;">50033</td>
			<td>4% Induction; 2.5% Maintenance</td>
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			<td height="40" style="height:40px;width:289px;">Isoflurane Vaporizer</td>
			<td style="width:187px;">Summit Medical Equipment</td>
			<td style="width:119px;">470062</td>
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			<td height="20" style="height:20px;">Stryker T/Pump Warm Water Recirculator</td>
			<td>Kent Scientific</td>
			<td>TP-700</td>
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			<td height="40" style="height:40px;width:289px;">Artifical Tears Lubricant Opthalmic Ointment</td>
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			<td>Norbrook Laboratories</td>
			<td colspan="2">NDC 55529-131-01</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>
			<td style="width:187px;">Fine Science Tools</td>
			<td style="width:119px;">17004-05</td>
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			<td height="20" style="height:20px;">Schwartz Micro Serrefines - Sharp Bend</td>
			<td>Fine&#160;Science Tools</td>
			<td>18052-03</td>
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			<td height="20" style="height:20px;">Surgical Instruments</td>
			<td>Fine Science Tools</td>
			<td></td>
			<td>sharp dissecting forceps, blunt forceps, fine scissors, spring scissors, hemostat</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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		<tr height="40">
			<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>
			<td></td>
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		<tr height="20">
			<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>
			<td></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 ...

The overall goal of this surgical procedure is to create a highly reproducible murine model of arterial denudation injury in the infrarenal abdominal aorta to examine arterial re-endothelialization. This method can help answer key questions in vascular biology. For example, what are the molecular mechanisms involved in endothelial regeneration, and what is the contribution endothelial cell progenitors in the process of repair of a wounded endothelium?This technique is a unique and practical tool for imposing denudation injury in mouse transgenic models. Gaining insight into the molecular mechanisms of re-endothelialization will aid in preventing thrombotic events following stent deployment in coronary artery and peripheral vascular disease. Though this method can be used to provide insight into the healing arterial denudation injury, it can also be used to investigate other methods of vascular injury repair.To begin this procedure, place the anesthetized mouse in the supine position on the heated surgical platform and cover its face with the nose cone. Then secure all the extremities with tape and carefully position it so that the abdomen is visible under the stereo microscope. After that, place the sterile adhesive dressings along each edge of the prepared surgical area.Now, make a three-centimeter incision down the midline of the animal's abdomen, using a scalpel, starting approximately 0.5 centimeters inferior to the xiphoid process. Gentry retract the skin with forceps and remove the skin from the abdominal wall by cutting the fine connective tissue. Apply 0.5%Bupivacaine to the muscle wall, then make a two-centimeter incision on the abdominal wall to expose the abdominal organs.After that, gently lift the intestines using saline-soaked cotton-tipped applicators, being careful to avoid blunt trauma to the jejunal and ilial arteries. Place them on a warm, sterile saline-soaked gauze sponge outside the abdominal cavity. Then cover the intestines with another warm, sterile saline-soaked gauze sponge to avoid moisture loss.Next, place a retractor to lateralize the rectum and expose the retroperitoneum. At the level of the inferior pole of the right kidney, use sharp dissecting forceps to make a retroperitonotomy lateral to the aorta, being careful not to injure the inferior vena cava or the surrounding vessels. Then, bluntly dissect the retroperitoneal tissue off the aorta.Place the vascular clamp over the aorta for at least one minute or other specified time and verify occlusion by visually observing the lack of pulsatility in the distal aorta. Following that, remove the vascular clamp and verify hemostasis. Confirm extravasation by adding 0.5 milliliters of saline into the abdominal cavity and assessing if the saline becomes increasingly blood-tinged.In this case, no extravasation is noted. Remove any gauze pads placed to aid in visualization of the retroperitoneum and place the intestines back into the abdomen. Subsequently, irrigate the abdominal cavity using pre-warmed sterile saline.In this procedure, close the abdominal wall muscle layer using a 5-0 braided absorbable single-running suture. Close the skin with one to three drops of polymer adhesive, and then close it with wound clips once the adhesive is set. Afterward, Carprofen is administered and the mouse is transferred to a recovery cage on a heating pad with food and water on the floor.Closely monitor the mouse for signs of respiratory distress, and administer Carprofen daily for 48 hours post-operatively. Do not leave the animal unattended until it has regained sufficient consciousness to maintain sternal recumbency. Subsequently, return the mouse to a normal cage with food and water.At any point after the surgery, the mouse can be sacrificed for evaluation. Using a dissecting stereo microscope, carefully separate the intact abdominal aorta from the surrounding tissues. Dissect the adherent connective tissue off the abdominal aorta, then open the aorta via a longitudinal incision along the dorsal surface.Subsequently, pin the aorta flat on a 35-millimeter silicone-coated dish with the luminal side up for fixation. Subsequently, the tissue can be either embedded for sectioning or used in onfas whole-mount immunocytochemistry. Here, histological evaluation of an uninjured aorta shows the intact tunica intima.In contrast, clamping of the aorta for ten seconds effectively removed the tunica intima with moderate damage to the smooth muscle cell layer, as demonstrated by H and E staining. Denudation of the endothelium occurs at both 10 seconds and 10 minutes, although to different degrees. To determine the optimal aortic clamping time interval required for complete arterial denudation, mice underwent clamping for 10 seconds, one minute, and 10 minutes, and were immediately sacrificed for analysis, as described in the protocol.The area of denudation increased with increasing clamp timings. We deemed one minute of aortic clamping sufficient, which produced a highly reproducible area of denudation measuring 0.88 square millimeters, on average, to result in near-complete arterial denudation injury without evidence of ischemic injury. Measurement of the fibrinogen staining 24 hours after injury of six aortas is approximately 0.81 square millimeters, statistically similar to the 0.88 square millimeter injury noted immediately after injury.Compared to the uninjured endothelium, the wound margin, 24 hours after injury, shows underlying fibrinogen staining, marking re-endothelialization into the denudation injury. Once mastered, this technique can be safely performed within 30 minutes, with ease. By varying the clamp type, application time and area, this technique offers tremendous versatility in denudation injury size that may be manipulated to fit one's study aims.Following this procedure, immunocytochemistry can be performed to suit one's study aims, such as investigating the proliferative potential of endothelial cells during re-endothelialization, or the contribution of inflammatory cells to vascular injury repair. After this development, this technique paved the way for researchers in the field of vascular biology to explore the role of re-endothelialization in vascular injury repair for transgenic mouse models. After watching this video, you should have a good understanding of how to create a reproducible murine model of arterial denudation injury in the infrarenal abdominal aorta.

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