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>
	<li>Farooq, V., Gogas, B. D., Serruys, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Restenosis:+delineating+the+numerous+causes+of+drug-eluting+stent+restenosis.">Restenosis: delineating the numerous causes of drug-eluting stent restenosis.</a> Circ Cardiovasc Interv. 4, 195-205 (2011).</li><li>Tada, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Risk+of+stent+thrombosis+among+bare-metal+stents,+first-generation+drug-eluting+stents,+and+second-generation+drug-eluting+stents:+results+from+a+registry+of+18,334+patients.">Risk of stent thrombosis among bare-metal stents, first-generation drug-eluting stents, and second-generation drug-eluting stents: results from a registry of 18,334 patients.</a> JACC Cardiovasc Interv. 6, 1267-1274 (2013).</li><li>Otsuka, F., 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=The+importance+of+the+endothelium+in+atherothrombosis+and+coronary+stenting.">The importance of the endothelium in atherothrombosis and coronary stenting.</a> Nat Rev Cardiol. 9, 439-453 (2012).</li><li>Kipshidze, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+the+endothelium+in+modulating+neointimal+formation:+vasculoprotective+approaches+to+attenuate+restenosis+after+percutaneous+coronary+interventions.">Role of the endothelium in modulating neointimal formation: vasculoprotective approaches to attenuate restenosis after percutaneous coronary interventions.</a> J Am Coll Cardiol. 44, 733-739 (2004).</li><li>Finn, A. V., 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=Pathological+correlates+of+late+drug-eluting+stent+thrombosis:+strut+coverage+as+a+marker+of+endothelialization.">Pathological correlates of late drug-eluting stent thrombosis: strut coverage as a marker of endothelialization.</a> Circulation. 115, 2435-2441 (2007).</li><li>Joner, 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=Pathology+of+drug-eluting+stents+in+humans:+delayed+healing+and+late+thrombotic+risk.">Pathology of drug-eluting stents in humans: delayed healing and late thrombotic risk.</a> J Am Coll Cardiol. 48, 193-202 (2006).</li><li>Joner, 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=Endothelial+cell+recovery+between+comparator+polymer-based+drug-eluting+stents.">Endothelial cell recovery between comparator polymer-based drug-eluting stents.</a> J Am Coll Cardiol. 52, 333-342 (2008).</li><li>McDonald, A. I., Iruela-Arispe, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Healing+arterial+ulcers:+Endothelial+lining+regeneration+upon+vascular+denudation+injury.">Healing arterial ulcers: Endothelial lining regeneration upon vascular denudation injury.</a> Vascul Pharmacol. 72, 9-15 (2015).</li><li>Fingerle, J., Tina Au, Y. P., Clowes, A. W., Reidy, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intimal+Lesion+Formation+in+Rat+Carotid+Arteries+after+Endothelial+Denudation+in+Absence+of+Medial+Injury.">Intimal Lesion Formation in Rat Carotid Arteries after Endothelial Denudation in Absence of Medial Injury.</a> Arteriosclerosis. 10, 1082-1087 (1990).</li><li>Granada, J. F., 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+response+to+zotarolimus-coated+balloons+in+injured+superficial+femoral+arteries+of+the+familial+hypercholesterolemic+Swine.">Vascular response to zotarolimus-coated balloons in injured superficial femoral arteries of the familial hypercholesterolemic Swine.</a> Circ Cardiovasc Interv. 4, 447-455 (2011).</li><li>Tulis, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rat+carotid+artery+balloon+injury+model.">Rat carotid artery balloon injury model.</a> Methods Mol Med. 139, 1-30 (2007).</li><li>Bavry, A. A., Bhatt, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Appropriate+use+of+drug-eluting+stents:+balancing+the+reduction+in+restenosis+with+the+concern+of+late+thrombosis.">Appropriate use of drug-eluting stents: balancing the reduction in restenosis with the concern of late thrombosis.</a> Lancet (London, England). 371, 2134-2143 (2008).</li><li>Gucu, 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=Effects+of+temporary+vascular+occluder+poloxamer+407+Gel+on+the+endothelium.">Effects of temporary vascular occluder poloxamer 407 Gel on the endothelium.</a> J Cardiothorac Surg. 8, (2013).</li><li>Holt, A. W., Tulis, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+Rat+and+Mouse+Carotid+Artery+Surgery:+Injury+&amp;+Remodeling+Studies.">Experimental Rat and Mouse Carotid Artery Surgery: Injury &amp; Remodeling Studies.</a> ISRN Minim Invasive Surg. 2013, (2013).</li><li>Nam, 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=Partial+carotid+ligation+is+a+model+of+acutely+induced+disturbed+flow,+leading+to+rapid+endothelial+dysfunction+and+atherosclerosis.">Partial carotid ligation is a model of acutely induced disturbed flow, leading to rapid endothelial dysfunction and atherosclerosis.</a> Am J Physiol Heart Circ Physiol. 297, 1535-1543 (2009).</li><li>Lindner, V., Fingler, J., Reidy, M. 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. Possible role of chronic vascular injury in restenosis.</a> Int J Cardiovasc Intervent. 2, 21-26 (1999).</li></ol>

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.

<table border="0" cellpadding="0" cellspacing="0" width="762">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="40">
			<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>
		</tr>
		<tr height="20">
			<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>
		</tr>
		<tr height="20">
			<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>
		</tr>
		<tr height="40">
			<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>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Stryker T/Pump Warm Water Recirculator</td>
			<td>Kent Scientific</td>
			<td>TP-700</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:289px;">Artifical Tears Lubricant Opthalmic Ointment</td>
			<td style="width:187px;">Akorn Animal Health</td>
			<td style="width:119px;">17478-162-35</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Carprieve (Carprofen)</td>
			<td>Norbrook Laboratories</td>
			<td colspan="2">NDC 55529-131-01</td>
		</tr>
		<tr height="20">
			<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>
		</tr>
		<tr height="20">
			<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>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Schwartz Micro Serrefines - Sharp Bend</td>
			<td>Fine&#160;Science Tools</td>
			<td>18052-03</td>
			<td></td>
		</tr>
		<tr height="20">
			<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>
		</tr>
		<tr height="20">
			<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>
			<td></td>
		</tr>
		<tr height="20">
			<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>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:289px;">Vetbond</td>
			<td style="width:187px;">Fisher Scientific</td>
			<td style="width:119px;">NC0304169</td>
			<td></td>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:289px;">Dissecting pins</td>
			<td style="width:187px;">Fisher Scientific</td>
			<td>NC9681411</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

reproducible-arterial-denudation-injury-infrarenal-abdominal-aortic

Research

JoVE Journal

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 ...

Reproducable Paraplegia by Thoracic Aortic Occlusion in a Murine Model of Spinal Cord Ischemia-reperfusion

Background 
Lower extremity paralysis continues to complicate aortic interventions. The lack of understanding of the underlying pathology has hindered advancements to decrease the occurrence this injury.&#160;The current model demonstrates reproducible lower&#160;extremity paralysis following thoracic aortic occlusion.
Methods 
Adult male C57BL6 mice were anesthetized with isoflurane. Through a cervicosternal incision the aorta was exposed. The descending thoracic aorta and left subclavian arteries were identified without entrance into pleural space. Skeletonization of these arteries was followed by immediate closure (Sham) or occlusion for 4 min (moderate ischemia) or 8 min (prolonged ischemia). The sternotomy and skin were closed and the mouse was transferred to warming bed for recovery.&#160; Following recovery, functional analysis was obtained at 12 hr intervals until 48 hr.
Results 
Mice that underwent sham surgery showed no observable hind&#160;limb deficit. Mice subjected to moderate ischemia for 4 min had minimal functional deficit at 12 hr followed by progression to complete paralysis at 48 hr. Mice subjected to prolonged ischemia had an immediate paralysis with no observable hind-limb movement at any point in the postoperative period. There was no observed intraoperative or post&#160;operative mortality.
Conclusion 
Reproducible lower extremity paralysis whether immediate or delayed can be achieved in a murine model. Additionally, by using a median sternotomy and careful dissection, high survival rates, and reproducibility can be achieved.

The lack of mechanistic understanding of spinal cord ischemia-reperfusion injury has hindered further adjuncts to prevent paraplegia following high risk aortic operations. Thus, the development of animal models is imperative. This manuscript demonstrates reproducible lower extremity paralysis following thoracic aortic occlusion in a murine model.

Background
Lower extremity paralysis continues to complicate aortic interventions. The lack of understanding of the underlying pathology has hindered ...

A Murine Model of Arterial Restenosis: Technical Aspects of Femoral Wire Injury

Cardiovascular disease caused by atherosclerosis is the leading cause of death in the developed world. Narrowing of the vessel lumen, due to atherosclerotic plaque development or the rupturing of established plaques, interrupts normal blood flow leading to various morbidities such as myocardial infarction and stroke. In the clinic endovascular procedures such as angioplasty are commonly performed to reopen the lumen. However, these treatments inevitably damage the vessel wall as well as the vascular endothelium, triggering an excessive healing response and the development of a neointimal plaque that extends into the lumen causing vessel restenosis (re-narrowing). Restenosis remains a major cause of failure of endovascular treatments for atherosclerosis. Thus, preclinical animal models of restenosis are vitally important for investigating the pathophysiological mechanisms as well as translational approaches to vascular interventions. Among several murine experimental models, femoral artery wire injury is widely accepted as the most suitable for studies of post-angioplasty restenosis because it closely resembles the angioplasty procedure that injures both endothelium and vessel wall. However, many researchers have difficulty utilizing this model due to its high degree of technical difficulty. This is primarily because a metal wire needs to be inserted into the femoral artery, which is approximately three times thinner than the wire, to generate sufficient injury to induce prominent neointima. Here, we describe the essential surgical details to effectively overcome the major technical difficulties of this model. By following the presented procedures, performing the mouse femoral artery wire injury becomes easier. Once familiarized, the whole procedure can be completed within 20 min.

The mouse femoral artery wire injury model of restenosis is technically challenging. In this protocol we show the key technical details essential for successfully performing wire injury to induce consistent&#160;neointima for studies of restenosis.

Cardiovascular disease caused by atherosclerosis is the leading cause of death in the developed world. Narrowing of the vessel lumen, due to ...

Open Tracheostomy Gastric Acid Aspiration Murine Model of Acute Lung Injury Results in Maximal Acute Nonlethal Lung Injury

Acid pneumonitis is a major cause of sterile acute lung injury (ALI) in humans. Acid pneumonitis spans the clinical spectrum from asymptomatic to acute respiratory distress syndrome (ARDS), characterized by neutrophilic alveolitis, and injury to both alveolar epithelium and vascular endothelium. Clinically, ARDS is defined by acute onset of hypoxemia, bilateral patchy pulmonary infiltrates and non-cardiogenic pulmonary edema. Human studies have provided us with valuable information about the physiological and inflammatory changes in the lung caused by ARDS, which has led to various hypotheses about the underling mechanisms. Unfortunately, difficulties determining the etiology of ARDS, as well as a wide range of pathophysiology have resulted in a lack of critical information that could be useful in developing therapeutic strategies.
Translational animal models are valuable when their pathogenesis and pathophysiology accurately reproduce a concept proven in both in vitro and clinical settings. Although large animal models (e.g., sheep) share characteristics of the anatomy of human trachea-bronchial tree, murine models provide a host of other advantages including: low cost; short reproductive cycle lending itself to greater data acquisition; a well understood immunologic system; and a well characterized genome leading to the availability of a variety of gene deletion and transgenic strains. A robust model of low pH induced ARDS requires a murine ALI that targets mainly the alveolar epithelium, secondarily the vascular endothelium, as well as the small airways leading to the alveoli. Furthermore, a reproducible injury with wide differences between different injurious and non-injurious insults is important.
The murine gastric acid aspiration model presented here using hydrochloric acid employs an open tracheostomy and recreates a pathogenic scenario that reproduces the low pH pneumonitis injury in humans. Additionally, this model can be used to examine interaction of a low pH insult with other pulmonary injurious entities (e.g., food particles, pathogenic bacteria).

This protocol induces acute lung injury in a mouse that has close fidelity to the pathogenesis of acid pneumonitis observed in humans. We generate a maximal acute nonlethal low pH lung injury and account for differences in rodent-human anatomic respiratory structure using an open tracheostomy coupled with circumferential pressure release.

Acid pneumonitis is a major cause of sterile acute lung injury (ALI) in humans. Acid pneumonitis spans the clinical spectrum from asymptomatic to acute ...

Creation of a Rodent Model of Abdominal Aortic Aneurysm by Blocking Adventitial Vasa Vasorum Perfusion

The adventitial vasa vasorum (VV) provides oxygen and nourishment to the aortic wall. Hypoxia in the aortic wall can cause enlarged abdominal aortic aneurysms (AAAs). This article introduces and describes a standard protocol used to induce AAAs through adventitial VV hypoperfusion created with a combination of polyurethane catheter insertion into the aortic lumen and suture ligation of the infrarenal abdominal aorta.
The protocol involves the use of male rats weighing 300-400 g, which are provided food and water ad libitum. After laparotomy with a ventral midline abdominal incision, exfoliation of the aorta is performed, which blocks blood flow from the perivascular tissue. Aortotomy involving a small incision adjacent to the renal artery branches is performed, and a polyurethane catheter is inserted using an 18-gauge indwelling needle. After repairing the incision, tight ligation of the aorta over the catheter blocks VV blood flow from the proximal direction through the aortic wall without disturbing the aortic blood flow. This technique can induce an AAA with progressive aortic dilatation.
The greatest benefit of this model is that VV hypoperfusion causes tissue hypoxia and the development of an infrarenal AAA, which has morphological and pathological characteristics similar to those of a human AAA.

Polyurethane catheter insertion into the aortic lumen and suture ligation of the aorta induce chronic hypoxia due to hypoperfusion of the adventitial vasa vasorum. This article describes a novel animal model of abdominal aortic aneurysm (AAA) with characteristics similar to those of AAA in humans.

The adventitial vasa vasorum (VV) provides oxygen and nourishment to the aortic wall. Hypoxia in the aortic wall can cause enlarged abdominal aortic ...

Reproducible Motor Deficit Following Aortic Occlusion in a Rat Model Of Spinal Cord Ischemia

Spinal cord ischemia is a fatal complication following thoracoabdominal aortic aneurysm surgery. Researchers can investigate the strategies for preventing and treating this complication using experimental models of spinal cord ischemia. The model described here demonstrates varying degrees of paraplegia that relate to the length of occlusion following thoracic aortic occlusion in a rat spinal cord ischemia model.
A 2-Fr. balloon-tipped catheter was advanced through the femoral artery into the descending thoracic aorta until the catheter tip was placed at the left subclavian artery in anesthetized male Sprague-Dawley rats. Spinal cord ischemia was induced by inflating the catheter balloon. After a set period of occlusion (9, 10, or 11 min), the balloon was deflated. Neurologic assessment was performed using the motor deficit index at 24 h after surgery, and the spinal cord was harvested for histopathological examination.
Rats that underwent 9 min of aortic occlusion showed mild and reversible motor impairment in the hind limb. Rats subjected to 10 min of aortic occlusion presented with moderate but reversible motor impairment. Rats subjected to 11 min of aortic occlusion displayed complete and persistent paralysis. The motor neurons in the spinal cord sections were more preserved in rats subjected to shorter duration of aortic occlusion.
Researchers can achieve a reproducible hind limb motor deficit following thoracic aortic occlusion using this spinal cord ischemia model.

This study demonstrates the technique to make a minimally invasive and easily reproducible model of spinal cord ischemia in rats. Various degrees of hind limb motor deficit can be produced by controlling the aortic occlusion time.

Spinal cord ischemia is a fatal complication following thoracoabdominal aortic aneurysm surgery. Researchers can investigate the strategies for preventing ...

Intravital Microscopy of Monocyte Homing and Tumor-Related Angiogenesis in a Murine Model of Peripheral Arterial Disease

The therapeutic goal for peripheral arterial disease and ischemic heart disease is to increase blood flow to ischemic areas caused by hemodynamic stenosis. Vascular surgery is a viable option in selected cases, but for patients without indications for surgery such as progression to rest pain, critical limb ischemia, or major disruptions to life or work, there are few possibilities for mitigating their disease. Cell therapy via monocyte-enhanced perfusion through the stimulation of collateral formation is one of a few non-invasive options.
Our group examines arteriogenesis after monocyte transplantation into mice using the hindlimb ischemia model. Previously, we have demonstrated improvement in hindlimb perfusion using tetanus-stimulated syngeneic monocyte transplantation. In addition to the effects on the collateral formation, tumor growth could be affected by this therapy as well. To investigate these effects, we use a basement membrane-like matrix mouse model by injecting the extracellular matrix of the Engelbreth-Holm-Swarm sarcoma into the flank of the mouse, after occlusion of the femoral artery.
After the artificial tumor studies, we use intravital microscopy to study in vivo tumor-angiogenesis and monocyte homing within collateral arteries. Previous studies have described the histological examination of animal models, which presupposes subsequent analysis to post-mortem artifacts. Our approach visualizes monocyte homing to areas of collateralization in real time sequences, is easy to perform, and investigates the process of arteriogenesis and tumor angiogenesis in vivo.

Monocytes are important mediators of arteriogenesis in the context of peripheral arterial disease. Using a basement membrane-like matrix and intravital microscopy, this protocol investigates monocyte homing and tumor-related angiogenesis after monocyte injection in the femoral artery ligation murine model.

The therapeutic goal for peripheral arterial disease and ischemic heart disease is to increase blood flow to ischemic areas caused by hemodynamic ...

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. ...

Porcine Model of Infrarenal Abdominal Aortic Aneurysm

Large animal models to study abdominal aortic aneurysms are sparse. The purpose of this model is to create reproducible, clinically significant infrarenal abdominal aortic aneurysms (AAA) in swine. To achieve this, we use a combination of balloon angioplasty, elastase and collagenase, and a lysyl oxidase inhibitor, called &#946;-aminopropionitrile (BAPN), to create clinically significant infrarenal aortic aneurysms, analogous to human disease.
Noncastrated male swine are fed BAPN for 7 days prior to surgery to achieve a steady state in the blood. A midline laparotomy is performed and the infrarenal aorta is circumferentially dissected. An initial measurement is recorded prior to aneurysm induction with a combination of balloon angioplasty, elastase (500 units)/collagenase (8000 units) perfusion, and topical elastase application. Swine are fed BAPN daily until terminal procedure on either postoperative day 7, 14, or 28, at which time the aneurysm is measured, and tissue procured. BAPN + surgery pigs are compared to pigs that underwent surgery alone.
Swine treated with BAPN and surgery had a mean aortic dilation of 89.9% &#177; 47.4% at day 7, 105.4% &#177; 58.1% at day 14, and 113.5% &#177; 30.2% at day 28. Pigs treated with surgery alone had significantly smaller aneurysms compared to BAPN + surgery animals at day 28 (p &#60; 0.0003). The BAPN + surgery group had macroscopic and immunohistochemical evidence of end stage aneurysmal disease.
Clinically significant infrarenal AAA can be induced using balloon angioplasty, elastase/collagenase perfusion and topical application, supplemented with oral BAPN. This model creates large, clinically significant AAA with hallmarks of human disease. This has important implications for the elucidation of AAA pathogenesis and testing of novel therapies and devices for the treatment of AAA. Limitations of the model include variation in BAPN ingested by swine, quality of elastase perfusion, and cost of BAPN.&#160;

This novel model creates robust infrarenal abdominal aortic aneurysms in swine using a combination of balloon angioplasty, elastase/collagenase perfusion, topical elastase application, and oral compound &#946;-aminopropionitrile administration, which interferes with collagen cross-linking.

Large animal models to study abdominal aortic aneurysms are sparse. The purpose of this model is to create reproducible, clinically significant infrarenal ...

A Murine Model of Ischemic Retinal Injury Induced by Transient Bilateral Common Carotid Artery Occlusion

Diverse vascular diseases such as diabetic retinopathy, occlusion of retinal veins or arteries and ocular ischemic syndrome can lead to retinal ischemia. To investigate pathological mechanisms of retinal ischemia, relevant experimental models need to be developed. Anatomically, a main retinal blood supplying vessel is the ophthalmic artery (OpA) and OpA originates from the internal carotid artery of the common carotid artery (CCA). Thus, disruption of CCA could effectively cause retinal ischemia. Here, we established a mouse model of retinal ischemia by transient bilateral common carotid artery occlusion (tBCCAO) to tie the right CCA with 6-0 silk sutures and to occlude the left CCA transiently for 2 seconds via a clamp, and showed that tBCCAO could induce acute retinal ischemia leading to retinal dysfunction. The current method reduces reliance on surgical instruments by only using surgical needles and a clamp, shortens occlusion time to minimize unexpected animal death, which is often seen in mouse models of middle cerebral artery occlusion, and maintains reproducibility of common retinal ischemic findings. The model can be utilized to investigate the pathophysiology of ischemic retinopathies in mice and further can be used for in vivo drug screening.

Here, we describe a mouse model of retinal ischemia by transient bilateral common carotid artery occlusion using simple sutures and a clamp. This model can be useful for understanding the pathological mechanisms of retinal ischemia caused by cardiovascular abnormalities.

Diverse vascular diseases such as diabetic retinopathy, occlusion of retinal veins or arteries and ocular ischemic syndrome can lead to retinal ischemia. ...