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.</li>
	<li>Place the mouse in the anesthesia induction chamber and induce with 4% isoflurane at a flow rate of 1 L/min.
	<ol>
		<li>Carefully monitor the respiratory rate and footpad skin color of mouse during induction.</li>
		<li>After slowing of the respiratory rate, remove the mouse from the induction chamber and place on warmed pad for surgical preparation with face in separate nosecone and maintenance isoflurane concentration of 2.5%.</li>
		<li>Perform toe pinch to assess adequacy of anesthetic.</li>
	</ol>
	</li>
	<li>Apply ophthalmic ointment to the corneas and administer preoperative carprofen (5 mg/kg) subcutaneously.</li>
	<li>Place mouse supine and use clippers to remove hair from abdomen.</li>
	<li>Prepare the surgical area with three alternating scrubs of povidone iodine and 70% isopropyl alcohol applied with sterile gauze pads.</li>
	<li>Place mouse supine on heated rodent surgical platform with face in nosecone and secure all extremities and carefully position mouse such that the abdomen is visible with the stereomicroscope.</li>
	<li>Place sterile, adhesive dressings along each edge of the prepared surgical area.</li>
	<li>Titrate isoflurane concentration to maintain adequate anesthesia during surgery while maintaining spontaneous respirations.</li>
</ol>
2. Infrarenal Aortic Clamping
<ol>
	<li>Make a 3 cm incision down the midline of the abdomen using a scalpel, starting approximately 0.5 cm inferior to the xiphoid process.</li>
	<li>Gently retract the skin with forceps and dissect skin away from the abdominal wall using fine scissors to cut the fine connective tissue.</li>
	<li>Apply 0.05-0.1 ml of 0.5% bupivacaine to the muscle wall and make a 2 cm incision into the abdominal wall to expose the abdominal organs.
	<ol>
		<li>If bleeding occurs along abdominal wall, apply gentle pressure with a cotton-tipped applicator.</li>
	</ol>
	</li>
	<li>Gently lift the intestines using saline-soaked cotton-tipped applicators. 
	NOTE: Be careful to avoid blunt trauma to the jejunal and ileal arteries and place on a warm, sterile saline soaked gauze sponge outside the abdominal cavity.
	<ol>
		<li>Cover the intestines with another warm, sterile saline soaked gauze sponge to avoid moisture loss.</li>
	</ol>
	</li>
	<li>Place a retractor to lateralize the rectum and expose the retroperitoneum.</li>
	<li>Place small gauze pads as necessary for visualization of retroperitoneum keeping track of the number used.</li>
	<li>At the level of the inferior pole of the right kidney, use sharp dissecting forceps to make a retroperitonotomy lateral to the aorta. 
	NOTE: Be careful not to injure the inferior vena cava or surrounding vessels.
	<ol>
		<li>Bluntly dissect retroperitoneal tissue off the aorta being careful not to perforate the inferior vena cava or surrounding vasculature.</li>
	</ol>
	</li>
	<li>Place the vascular clamp over the aorta for at least 1 min, or other specified time, and verify occlusion by visually observing lack of pulsatility in distal aorta.</li>
	<li>Remove the vascular clamp and verify hemostasis. Hemostasis is achieved and ensured when no active extravasation of blood is seen.
	<ol>
		<li>Confirm extravasation by adding 0.5 ml saline into abdominal cavity and assessing if the saline becomes increasingly blood-tinged. If this is the case, apply gentle pressure using the saline-soaked applicator for 1 min to ensure hemostasis.</li>
	</ol>
	</li>
	<li>Remove any gauze pads placed to aid in visualization of retroperitoneum and replace intestines in situ into abdomen.</li>
	<li>Irrigate the abdominal cavity using pre-warmed sterile saline.</li>
</ol>
3. Closure of Laparotomy and Skin
<ol>
	<li>Close the abdominal wall muscle layer using a 5-0 braided, absorbable single running suture.</li>
	<li>Close skin with 1-3 drops of polymer adhesive and then subsequently with wound clips once the adhesive is set.</li>
</ol>
4. Recovery and Postoperative Assessment
<ol>
	<li>Transfer the mouse to a recovery cage on a heating pad with food and water on the floor of the cage.</li>
	<li>Closely monitor the mouse for signs of respiratory distress. Administer carprofen (5 mg/kg) daily for 48 hr postoperatively per institution guidelines.
	<ol>
		<li>Do not leave an animal unattended until it has regained sufficient consciousness to maintain sternal recumbency.</li>
	</ol>
	</li>
	<li>Return the mouse to a normal cage with food and water.
	<ol>
		<li>Do not return an animal that has undergone surgery to the company of other animals until fully recovered.</li>
	</ol>
	</li>
	<li>Assess wound on a daily basis for dehiscence and remove clips on postoperative day 14.</li>
</ol>
5. Aortic Dissection and Staining
<ol>
	<li>Select mice for sacrifice based on the specific time point of interest following denudation injury.</li>
	<li>Sacrifice mice via isoflurane inhalation and immediately inject with 5 mg methacholine chloride to cause vascular smooth muscle relaxation.</li>
	<li>Upon confirmation of death by respiratory cessation, lack of corneal reflex and lack of movement, perfuse with 4% paraformaldehyde in phosphate buffered saline at a perfusion pressure of 100 mmHg via the left heart ventricle for 10 min.</li>
	<li>Use a dissecting stereomicroscope to carefully separate the intact abdominal aorta from the surrounding tissues.</li>
	<li>Transect the aorta rostral to the renal arteries and caudal to the iliac bifurcation and opened via a longitudinal incision along the dorsal surface.</li>
	<li>Pin the aorta flat on a 35 mm silicone coated dish with the luminal side up for fixation for no less than 2 hr but no more than 12 hr. Subsequently, the tissue can be either embedded for sectioning or used in en face whole-mount immunocytochemistry.</li>
	<li>Mount stained aortas with luminal side facing the coverslip on glass slides for confocal microscopy.</li>
</ol>

<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. 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The authors have nothing to disclose.

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><tbody><tr><td>Zeiss Discovery.V12 Stereomicroscope</td><td>Zeiss</td><td>495037-9904-000</td><td></td></tr><tr><td>Rodent Heated Surgical Platform</td><td>Protech International</td><td>RES4000</td><td>Heated platform for body temperature maintenance with nosecone for anesthestic maintenance and on which surgical procedure is performed</td></tr><tr><td>Isoflurane</td><td>Henry Schein</td><td>50033</td><td>4% Induction; 2.5% Maintenance</td></tr><tr><td>Isoflurane Vaporizer</td><td>Summit Medical Equipment</td><td>470062</td><td></td></tr><tr><td>Stryker T/Pump Warm Water Recirculator</td><td>Kent Scientific</td><td>TP-700</td><td></td></tr><tr><td>Artifical Tears Lubricant Opthalmic Ointment</td><td>Akorn Animal Health</td><td>17478-162-35</td><td></td></tr><tr><td>Carprieve (Carprofen)</td><td>Norbrook Laboratories</td><td>NDC 55529-131-01</td><td></td></tr><tr><td>Oster&amp;trade; A5 Professional Animal Clipper</td><td>M.Schneider &amp;amp; Sons Inc.</td><td>78005010</td><td>Use with animal clipper size 40</td></tr><tr><td>Adjustable Wire Retractor</td><td>Fine Science Tools</td><td>17004-05</td><td></td></tr><tr><td>Schwartz Micro Serrefines - Sharp Bend</td><td>Fine&amp;nbsp;Science Tools</td><td>18052-03</td><td></td></tr><tr><td>Surgical Instruments</td><td>Fine Science Tools</td><td></td><td>sharp dissecting forceps, blunt forceps, fine scissors, spring scissors, hemostat</td></tr><tr><td>0.5% Marcaine</td><td>Hospira</td><td>0409-1610-50</td><td></td></tr><tr><td>5-0 Suture, Vicryl</td><td>Fisher Scientific</td><td>NC0189890</td><td>tapered needle</td></tr><tr><td>Vetbond</td><td>Fisher Scientific</td><td>NC0304169</td><td></td></tr><tr><td>Falcon&amp;reg; 35 mm Not TC-Treated Easy-Grip Style Bacteriological Petri Dish</td><td>Corning</td><td>351008</td><td></td></tr><tr><td>Sylgard 184 Silicone Elastomer Kit</td><td>Dow Corning</td><td>3097358-1004</td><td></td></tr><tr><td>Dissecting pins</td><td>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 ...

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9.2K Views.  University of California, Los Angeles.  Het begrijpen van de cellulaire en moleculaire mechanismen van re-endothelialisatie na arteriële ontbloting letsel is van het grootste belang bij het voorkomen van trombose en restenose van slagaders. Hier beschrijven we een protocol voor reproduceerbare arteriële erosie letsel van de infrarenale abdominale aorta. De procedure werd ontwikkeld om de onderliggende mechanismen die endotheliale regeneratie met behulp van muismodellen te regelen onderzoeken. 

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

Use of a Hanging-weight System for Isolated Renal Artery Occlusion

In hospitalized patients, over 50% of cases of acute kidney injury (AKI) are caused by renal ischemia 1-3. A recent study of hospitalized patients revealed that only a mild increase in serum creatinine levels (0.3 to 0.4 mg/dl) is associated with a 70% greater risk of death than in persons without any increase 1. Along these lines, surgical procedures requiring cross-clamping of the aorta and renal vessels are associated with a renal failure rates of up to 30% 4. Similarly, AKI after cardiac surgery occurs in over 10% of patients under normal circumstances and is associated with dramatic increases in mortality. AKI are also common complications after liver transplantation. At least 8-17% of patients end up requiring renal replacement therapy 5. Moreover, delayed graft function due to tubule cell injury during kidney transplantation is frequently related to ischemia-associated AKI 6. Moreover, AKI occurs in approximately 20% of patients suffering from sepsis 6.The occurrence of AKI is associated with dramatic increases of morbidity and mortality 1. Therapeutic approaches are very limited and the majority of interventional trials in AKI have failed in humans. Therefore, additional therapeutic modalities to prevent renal injury from ischemia are urgently needed 3, 7-9. 
To elucidate mechanisms of renal injury due to ischemia and possible therapeutic strategies murine models are intensively required 7-13. Mouse models provide the possibility of utilizing different genetic models including gene-targeted mice and tissue specific gene-targeted mice (cre-flox system). However, murine renal ischemia is technically challenging and experimental details significantly influence results. We performed a systematic evaluation of a novel model for isolated renal artery occlusion in mice, which specifically avoids the use of clamping or suturing the renal pedicle 14. This model requires a nephrectomy of the right kidney since ischemia can be only performed in one kidney due to the experimental setting. In fact, by using a hanging-weight system, the renal artery is only instrumented once throughout the surgical procedure. In addition, no venous or urethral obstruction occurs with this technique. We could demonstrate time-dose-dependent and highly reproducible renal injury with ischemia by measuring serum creatinine. Moreover, when comparing this new model with conventional clamping of the whole pedicle, renal protection by ischemic preconditioning is more profound and more reliable. Therefore his new technique might be useful for other researchers who are working in the field of acute kidney injury.

A precise murine model for acute kidney injury (AKI) due to ischemia is an important tool to investigate acute kidney injury and possibly find therapeutic tools to treat renal injury. The hanging weight system offers a tool for immediate and reliable renal artery occlusion and reperfusion without causing renal congestion.

In hospitalized patients, over 50% of cases of acute kidney injury (AKI) are caused by renal ischemia 1-3. A recent study of hospitalized patients ...

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 ...

Murine Model of Femoral Artery Wire Injury with Implantation of a Perivascular Drug Delivery Patch

Percutaneous interventions including balloon angioplasty and stenting have been used to restore blood flow in vessels with occlusive vascular disease. While these therapies lead to the rapid restoration of blood flow, these technologies remain limited by restenosis in the case of bare metal stents and angioplasty, or reduced healing and possibly enhanced risk of thrombosis in the case of drug eluting stents. A key pathophysiological mechanism in the formation of restenosis is intimal hyperplasia caused by the activation of vascular smooth muscle cells and inflammation due to arterial stretch and injury. Surgeries that induce arterial injury in genetically modified mice are useful for the mechanistic study of the vascular response to injury but are often technically challenging to perform in mouse models due to the their small size and lack of appropriate sized devices. We describe two approaches for a surgical technique that induces endothelial denudation and arterial stretch in the femoral artery of mice to produce robust neointimal hyperplasia. The first approach creates an arteriotomy in the muscular branch of the femoral artery to obtain vascular access. Following wire injury this arterial branch is ligated to close the arteriotomy. A second approach creates an arteriotomy in the main femoral artery that is later closed through localized cautery. This method allows for vascular access through a larger vessel and, consequently, provides a less technically demanding procedure that can be used in smaller mice. Following either method of arterial injury, a degradable drug delivery patch can be placed over or around the injured artery to deliver therapeutic agents.

We describe a surgical technique that produces wire injury in the femoral artery of mice to induce neointimal hyperplasia to serve as a model testing system for the perivascular delivery of therapeutic compounds for the inhibition of restenosis.

Percutaneous interventions including balloon angioplasty and stenting have been used to restore blood flow in vessels with occlusive vascular disease. ...

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 ...

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 ...

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. ...

Murine Surgical Model of Topical Elastase Induced Descending Thoracic Aortic Aneurysm

According to the Center for Disease Control, aortic aneurysms (AAs) were considered a leading cause of death in all races and both sexes from 1999-2016. An aneurysm forms as a result of progressive weakening and eventual dilation of the aorta, which can rupture or tear once it reaches a critical diameter. Aneurysms of the descending aorta in the chest, called descending thoracic aortic aneurysms (dTAA), make up a large proportion of aneurysm cases in the United States. Uncontained dTAA rupture is almost universally lethal, and elective repair has a high rate of morbidity and mortality. The purpose of our model is to study dTAA specifically, to elucidate the pathophysiology of dTAA and to search for molecular targets to halt the growth or reduce the size of dTAA. By having a murine model to study thoracic pathology precisely, targeted therapies can be developed to specifically test dTAA. The method is based on the placement of porcine pancreatic elastase (PPE) directly on the outer murine aortic wall after surgical exposure. This creates a destructive and inflammatory reaction, which weakens the aortic wall and allows for aneurysm formation over weeks to months. Though murine models possess limitations, our dTAA model produces robust aneurysms of predictable size. Furthermore, this model can be used to test genetic and pharmaceutical targets which may arrest dTAA growth or prevent rupture. In human patients, interventions such as these could help avoid aneurysm rupture, and difficult surgical intervention.

We describe a surgical protocol to consistently induce robust descending thoracic aortic aneurysms in mice. The procedure involves left thoracotomy, thoracic aorta exposure, and placement of a sponge soaked in porcine pancreatic elastase on the aortic wall.

According to the Center for Disease Control, aortic aneurysms (AAs) were considered a leading cause of death in all races and both sexes from 1999-2016. ...

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 ...

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.

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.

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

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

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