Name: the rabbit model of accelerated atherosclerosis: a methodological perspective of the iliac artery balloon injury
Uploaded: 2017-10-03T12:30:00
Description: Watch this Scientific Journal Video about The Rabbit Model of Accelerated Atherosclerosis: A Methodological Perspective of the Iliac Artery Balloon Injury at JoVE.com

This work was supported by the Swiss National Science Foundation Grant 150271.

This experimental protocol has been approved by the Cantonal Veterinary Office, Fribourg and the Swiss Federal Veterinary Office, Switzerland (FR 2015/58).
NOTE: Male NZW rabbits weighing between 2.8 to 3.2 kg were used. The animals were housed under conventional conditions (12 h light and dark cycle, provided ad libitum water and food). Prior to balloon denudation, animals were acclimated for 1 week during which they were fed with normal chow diet. After 1 week of acclimatization, rabbits wer...

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rat+carotid+artery+balloon+injury+model.">Rat carotid artery balloon injury model.</a> Methods Mol Med. 139, 1-30 (2007).</li><li>Asada, Y., 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+inflation+pressure+of+balloon+catheter+on+vascular+injuries+and+subsequent+development+of+intimal+hyperplasia+in+rabbit+aorta.">Effects of inflation pressure of balloon catheter on vascular injuries and subsequent development of intimal hyperplasia in rabbit aorta.</a> Atherosclerosis. 121, 45-53 (1996).</li><li>Dornas, W. C., Oliveira, T. T., Augusto, L. E., Nagem, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+atherosclerosis+in+rabbits.">Experimental atherosclerosis in rabbits.</a> Arq Bras Cardiol. 95, 272-278 (2010).</li><li>Waksman, R., 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=PhotoPoint+photodynamic+therapy+promotes+stabilization+of+atherosclerotic+plaques+and+inhibits+plaque+progression.">PhotoPoint photodynamic therapy promotes stabilization of atherosclerotic plaques and inhibits plaque progression.</a> J Am Coll Cardiol. 52, 1024-1032 (2008).</li><li>Fernandez-Parra, R., 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=Pharmacokinetic+Study+of+Paclitaxel+Concentration+after+Drug-Eluting+Balloon+Angioplasty+in+the+Iliac+Artery+of+Healthy+and+Atherosclerotic+Rabbit+Models.">Pharmacokinetic Study of Paclitaxel Concentration after Drug-Eluting Balloon Angioplasty in the Iliac Artery of Healthy and Atherosclerotic Rabbit Models.</a> J Vasc Interv Radiol. 26, 1380-1387 (2015).</li><li>Dussault, S., Dhahri, W., Desjarlais, M., Mathieu, R., Rivard, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elsibucol+inhibits+atherosclerosis+following+arterial+injury:+multifunctional+effects+on+cholesterol+levels,+oxidative+stress+and+inflammation.">Elsibucol inhibits atherosclerosis following arterial injury: multifunctional effects on cholesterol levels, oxidative stress and inflammation.</a> Atherosclerosis. 237, 194-199 (2014).</li><li>Manderson, J. A., Mosse, P. R., Safstrom, J. A., Young, S. B., Campbell, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Balloon+catheter+injury+to+rabbit+carotid+artery.+I.+Changes+in+smooth+muscle+phenotype.">Balloon catheter injury to rabbit carotid artery. I. Changes in smooth muscle phenotype.</a> Arteriosclerosis. 9, 289-298 (1989).</li><li>Miyake, 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=Prevention+of+neointimal+formation+after+angioplasty+using+nuclear+factor-kappaB+decoy+oligodeoxynucleotide-coated+balloon+catheter+in+rabbit+model.">Prevention of neointimal formation after angioplasty using nuclear factor-kappaB decoy oligodeoxynucleotide-coated balloon catheter in rabbit model.</a> Circ Cardiovasc Interv. 7, 787-796 (2014).</li><li>Fulcher, J., Patel, S., Nicholls, S. 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The rabbit iliac artery atherosclerosis model is widely used in atherosclerosis research. With this protocol the rabbits rapidly developed more severe and advanced plaques as compared to spontaneous lesions developed with only cholesterol diet. Importantly, animals recover quickly from the surgery.
The main stimulus for atherogenesis is the mechanical damage caused by the balloon catheter that injures the endothelium and distends the vessel wall26. This procedure induce...

Rupture of vulnerable atherosclerotic plaques is one of the leading causes of death in the industrialized nations1. Although research over the past decades has unfolded several molecular and cellular mechanisms involved in plaque progression, continued efforts are still needed not only to unravel the complex mechanism of disease progression but also to test new therapeutic approaches. Several animal models have been proposed to study the atherosclerosis. Genetic manipulation, cholesterol feeding or mechanical endothelium injury are the standard strategies shared by most animal models of atherosclerosis including mice, rabbits or minipigs. Among these, NZW rabbits are sensitive to cholesterol diet while normal rats and mice do not significantly absorb dietary cholesterol2,3,4. Rabbits spontaneously develop aortic lesions rich in macrophages with some fibrous component when fed with cholesterol rich diet5,6. However, the long preparatory time of 4-8 months to induce atherosclerotic plaquesby feeding cholesterol diet alone6,7 is a major drawback for most of the experimental settings. In pursuit for inducing lesions in relatively short time, a combination of high cholesterol diet and balloon injury has been developed by Baumgarter and Studer8. The overall goal of this technique is to induce atherosclerotic plaques composed of foam cells (similar to fatty streak in humans) in hypercholesterolemic rabbits within 2 weeks. The present technique describes the procedure of arterial wall injury based on Baumgarter&#39;s method using a balloon catheter advanced into the iliac artery of NZW hypercholesterolemic rabbits.
Together with a cholesterol rich diet, injury resulting from balloon induced de-endothelialization will lead to atherosclerosis. Balloon injury accelerates the formation of atherosclerotic lesions, and produces plaque of uniform size and distribution. Intimal thickening increases over a period of time and intimal cell infiltration starts within few days following injury. Fatty streaks with substantial macrophages start to appear after 7 - 10 days of balloon injury and are represented as Type II lesion according to the classification by American Heart Association. Balloon injury in rabbit is often performed in the aorta to study plaque composition. The neointimal endothelium expresses high levels of intercellular adhesion molecule. The plaques are associated with medial dissection and adventitial changes. Atherosclerotic lesions are composed of lipids, proliferating smooth muscle cells (SMCs), collagen fibers and inflammatory cells that accumulate under the regenerated endothelium and are mostly type II in nature. The topological distribution of rabbit plaques was similar to that reported in human aortas 9,10 In principle, the aorta is larger in size compared to iliac arteries and would produce plaque in larger length. However, the major advantage of using the iliac artery as the site of atherosclerosis in rabbits is its accessibility, its similarity in muscular content to human coronary artery11, uniform lesion development12, high tissue factor activity13 and consistent vessel dimension comparable to human coronary artery allowing the evaluation of commercially manufactured devices to morphometric and angiographic endpoints. Invasive and non-invasive methods have been investigated to analyze the plaques in rabbit iliac arteries in the live animal. Previous reports describe the use of magnetic resonance imaging (MRI) with the help of a 2.35-tesla MR system 14 Additionally, intravascular ultrasound (IVUS) or optical coherence tomography (OCT) catheters can be suitably applied to image atherosclerotic plaques in rabbit iliac arteries. The iliac artery is accessible for ultrasound imaging when using a high-resolution echography and the aorta can also be explored with this technique.
In the past decade, this rabbit model of balloon injury has helped to further understand the mechanisms of plaque progression15and plaque regression16. In addition, the model has been used to study the influence of novel therapeutic agents such as statins, standard antiplatelet agents, antioxidant agents17,18 and drug-eluting stents such as everolimus or zotarolimus-eluting stent19,20 on neointimal thickening. This model has also been used to investigate intravascular imaging of near-infrared fluorescence imaging catheter21.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>New Zealand White rabbits</td>
			<td>Charles River laboratories,France</td>
			<td>Cre:KBL(NZW)</td>
			<td></td>
		</tr>
		<tr>
			<td>Cholesterol rich diet</td>
			<td>Ssniff spezialdi&#228;ten</td>
			<td>Ssniff EF K High Fat and Cholesterol</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass bead sterilizer-Germinator 500</td>
			<td>VWR, Leicestershire, UK</td>
			<td>101326-488</td>
			<td></td>
		</tr>
		<tr>
			<td>Fogarty balloon embolectomy catheters, 2 French</td>
			<td>Edwards Lifesciences, Switzerland</td>
			<td>120602F</td>
			<td>For single use only</td>
		</tr>
		<tr>
			<td>Luer Lock Syringe</td>
			<td>Becton, Dickinson and Company, USA</td>
			<td>309628</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermopad Type 226</td>
			<td>Solis, Switzerland AG</td>
			<td>397387</td>
			<td></td>
		</tr>
		<tr>
			<td>Buprenorphine- Temgesic</td>
			<td>Reckitt Benckiser AG, Switzerland</td>
			<td>7.68042E+12</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Piramal Critical Care, Inc, Bethlehem, PA 18017</td>
			<td>2667-46-7</td>
			<td></td>
		</tr>
		<tr>
			<td>Anaesthesia machine-combi-vet Base Anesthesia System</td>
			<td>Rothacher Medical GmbH, Switzerland</td>
			<td>CV 30-301-A</td>
			<td></td>
		</tr>
		<tr>
			<td>Cardell touch veterinary vital signs monitor</td>
			<td>Midmark, Ohio, USA</td>
			<td>8013-001</td>
			<td></td>
		</tr>
		<tr>
			<td>Ophthalmic ointment-Humigel</td>
			<td>Virbac, France</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Animal hair clippers</td>
			<td>Aesculap AG, Germany</td>
			<td>GT420</td>
			<td></td>
		</tr>
		<tr>
			<td>Disinfectant-Betadine solution</td>
			<td>MundipharmaMedicalCompany, Switzerland</td>
			<td>14671-1203</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont #7 Forceps</td>
			<td>FST Germany</td>
			<td>11274-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Medium and small microscissors</td>
			<td>Medline International Switzerland S&#224;rl</td>
			<td>UC4337</td>
			<td></td>
		</tr>
		<tr>
			<td>Microvascular clamps</td>
			<td>FST, Germany</td>
			<td>18051-28</td>
			<td></td>
		</tr>
		<tr>
			<td>Papaverine</td>
			<td>ESCA chemicals, Switzerland</td>
			<td>RE 356 803</td>
			<td></td>
		</tr>
		<tr>
			<td>Vein Pick</td>
			<td>Harvard Apparatus, Cambridge, UK</td>
			<td>72-4169</td>
			<td>For single use only</td>
		</tr>
		<tr>
			<td>Saline</td>
			<td>Laboratorium Dr. G. Bichsel AG, , Switzerland</td>
			<td>1330055</td>
			<td></td>
		</tr>
		<tr>
			<td>Polysorb 5-0 suture</td>
			<td>Covidien AG, Switzerland</td>
			<td>UL 202</td>
			<td>Monofilament</td>
		</tr>
		<tr>
			<td>Sulfadoxine and Trimethoprim-Trimethazol</td>
			<td>Werner Stricker AG, Switzerland</td>
			<td>Swissmedic Nr. 50&#39;361</td>
			<td></td>
		</tr>
		<tr>
			<td>Antiseptic- Octenisept</td>
			<td>Sch&#252;lke &#38; Mayr AG, Switzerland</td>
			<td>GTIN: 4032651214068</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline</td>
			<td>Roth</td>
			<td>1058.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Isobutanol-2-Methylbutane</td>
			<td>Sigma-Aldrich, Switzerland</td>
			<td>M32631-1L</td>
			<td></td>
		</tr>
		<tr>
			<td>Optimum Cutting Temperature compound-Tissue-Tek</td>
			<td>VWR Chemicals, Belgium</td>
			<td>25608-930</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryostat</td>
			<td>Leica, Glattbrugg, Switzerland</td>
			<td>Leica CM1860 UV</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass slide- Superfrost Plus</td>
			<td>Thermo Scientific</td>
			<td>4951PLUS4</td>
			<td></td>
		</tr>
		<tr>
			<td>Mayer&#39;s Haematoxylin</td>
			<td>Sigma-Aldrich, Switzerland</td>
			<td>MHS32-1L</td>
			<td></td>
		</tr>
		<tr>
			<td>Eosin 0.5% aq.</td>
			<td>Sigma-Aldrich, Switzerland</td>
			<td>HT110232-1L</td>
			<td></td>
		</tr>
		<tr>
			<td>Oil Red O</td>
			<td>Sigma-Aldrich, Switzerland</td>
			<td>O0625-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>&#945;-smooth muscle actin antibody</td>
			<td>Abcam, UK.</td>
			<td>ab7817</td>
			<td></td>
		</tr>
		<tr>
			<td>Macrophage Clone RAM11 antibody</td>
			<td>DAKO, Switzerland</td>
			<td>M063301</td>
			<td></td>
		</tr>
		<tr>
			<td>Hoechst</td>
			<td>Abcam, UK.</td>
			<td>ab145596</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat polyclonal Secondary Antibody (Chromeo 546)</td>
			<td>Abcam, UK.</td>
			<td>ab60316</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 488/547</td>
			<td>Abcam, UK.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glycergel Mounting Medium, Aqueous</td>
			<td>DAKO, Switzerland</td>
			<td>C056330</td>
			<td></td>
		</tr>
		<tr>
			<td>Hematoxylin for Movat pentachrome staining</td>
			<td>Sigma-Aldrich, Switzerland</td>
			<td>H3136-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>Ferric chloride for Movat pentachrome staining</td>
			<td>Sigma-Aldrich, Switzerland</td>
			<td>157740-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Iodine for Movat staining</td>
			<td>Sigma-Aldrich, Switzerland</td>
			<td>207772-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium iodide for Movat pentachrome staining</td>
			<td>Sigma-Aldrich, Switzerland</td>
			<td>60400-100G-F</td>
			<td></td>
		</tr>
		<tr>
			<td>Alcian blue for Movat staining</td>
			<td>Sigma-Aldrich, Switzerland</td>
			<td>A5268-10G</td>
			<td></td>
		</tr>
		<tr>
			<td>Strong Ammonia for Movat pentachrome staining</td>
			<td>Sigma-Aldrich, Switzerland</td>
			<td>320145-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Brilliant crocein MOO for Movat pentachrome staining</td>
			<td>Sigma-Aldrich, Switzerland</td>
			<td>210757-50G</td>
			<td></td>
		</tr>
		<tr>
			<td>Acid Fuchsin for Movat pentachrome staining</td>
			<td>Sigma-Aldrich, Switzerland</td>
			<td>F8129-50G</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Thiosulfate for Movat pentachrome staining</td>
			<td>Sigma-Aldrich, Switzerland</td>
			<td>72049-250G,</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphotungstic acid for Movat pentachrome staining</td>
			<td>Sigma-Aldrich, Switzerland</td>
			<td>79690-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Crocin for Movat pentachrome staining</td>
			<td>Sigma-Aldrich, Switzerland</td>
			<td>17304-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>EUKITT for Movat pentachrome staining</td>
			<td>Sigma-Aldrich, Switzerland</td>
			<td>03989-100ML</td>
			<td></td>
		</tr>
	</tbody>
</table>

the rabbit model of accelerated atherosclerosis: a methodological perspective of the iliac artery balloon injury

Acute coronary syndrome resulting from coronary occlusion following atherosclerotic plaque development and rupture is the leading cause of death in the industrialized world. New Zealand White (NZW) rabbits are widely used as an animal model for the study of atherosclerosis. They develop spontaneous lesions when fed with atherogenic diet; however, this requires long time of 4 - 8 months. To further enhance and accelerate atherogenesis, a combination of atherogenic diet and mechanical endothelial injury is often employed. The presented procedure for inducing atherosclerotic plaques in rabbits uses a balloon catheter to disrupt the endothelium in the left iliac artery of NZW rabbits fed with atherogenic diet. Such mechanical damage caused by the balloon catheter induces a chain of inflammatory reactions initiating neointimal lipid accumulation in a time dependent fashion. Atherosclerotic plaque following balloon injury show neointimal thickening with extensive lipid infiltration, high smooth muscle cell content and presence of macrophage derived foam cells. This technique is simple, reproducible and produces plaque of controlled length within the iliac artery. The whole procedure is completed within 20 - 30 min. The procedure is safe with low mortality and also offers high success in obtaining substantial intimal lesions. The procedure of balloon catheter induced arterial injury results in atherosclerosis within two weeks. This model can be used for investigating the disease pathology, diagnostic imaging and to evaluate new therapeutic strategies.

Balloon injury of the iliac artery was performed successfully without complication (Figure 1). The total operative time ranged from 20 to 30 min for injuries performed on only one iliac artery, and 35 to 45 min for injuries on both arteries. The rabbit recovered within 1 h after balloon injury. All animals appeared healthy without significant weight loss. No infection, oedema or arterial thrombosis was encountered. The wound area was normal besides some mild ...

Watch this Scientific Journal Video about The Rabbit Model of Accelerated Atherosclerosis: A Methodological Perspective of the Iliac Artery Balloon Injury at JoVE.com

The Rabbit Model of Accelerated Atherosclerosis: A Methodological Perspective of the Iliac Artery Balloon Injury

Animal models of atherosclerosis are essential to understand the mechanism and to investigate newer approaches to prevent plaque development or rupture, a leading cause of death in the industrialized world. This protocol uses a combination of balloon injury and cholesterol rich diet to induce atherosclerotic plaques in rabbit iliac artery.

Acute coronary syndrome resulting from coronary occlusion following atherosclerotic plaque development and rupture is the leading cause of death in the ...

The overall goal of this procedure is to induce the formation of atherosclerotic plaques in rabbits by surgically disrupting the endothelium of their left iliac arteries, and feeding them an atherogenic diet. By lone injury in rabbits is often performed in the aorta to study plaque composition. However, the iliac artery is very similar to the human coronary artery, making it an attractive place for modeling atherosclerotic plaques.The muscular content uniform lesions, eye tissue factor activity, and consistent vessel dimensions of the rabbit iliac artery are all comparable to the human coronary artery. Ultimately, this allows for the evaluation of commercially manufactured devices to morphometric and angiographic endpoints. Demonstrating the procedure will be Aurelien Frobert, a collaborator from my laboratory.Be certain to sterilize all the surgical tools using, for example, a glass bead sterilizer. Next, prepare and check the two balloon catheter assemblies. Onto each one, attach a one-milliliter Luer lock syringe filled with normal saline, and then check for trapped embliques using the balloon's inflation mechanism.Next, set the thermal pad at the surgical platform to 37 degree celsius. Then weigh the rabbit. Next, provide an injection of buprenorphine and anesthetize the rabbit using 5%isofluorane at five liters per minute for 10 to 15 minutes.Now, place the anesthetized rabbit on the heating pad on a surgical platform. Attach sensors to monitor the rabbit's temperature and respiration and to collect electrocardiograms. Next, attach a face mask to deliver 4%isofluorane at 2.5 liters per minute.Then confirm a proper level of anesthesia by testing for a lost of gag and petty reflexes. A universally relaxed muscle tone is also indicator of deep anesthesia. Next, apply abtonic ointment to both eyes.Next, remove the hair from the ventral area just below the knee joints using animal hair clippers. Then, remove the loose hair and clean the exposed skin with three alternating scrubs of 70%ethanol and betadine and drape the animal leaving only the lower limb exposed. To begin the surgery, locate the saphenous artery and make a 1.5 centimeter incision.Then using small curved forceps carefully expose a small portion of saphenous artery. Be careful to avoid damaging the femoral vein or femoral nerve. Cleaning the artery thoroughly is very important during this exposure.Next, place two loose ligature loops beneath the saphenous artery and tie one ligature loop near the distal end of the artery. Then place some micro vascular clamp along the artery above the ligature and close the clamp to stop the blood flow. Immediately apply a drop of papaverine to dilate the artery and prevent phase spasms.Next, using the ligature, lift the saphenous artery and to make a small arteriotomy incision using a 24 gauge needle. Then lift the incision flaps and slowly insert a vein pick or a guiding needle into the lumen of the artery. Now, carefully insert an arterial embolectomy catheter into the saphenous artery.Be gentle to avoid making ruptures. Then remove the vein pick and release the micro vascular clamp on the artery. To create the injury, first advance the balloon catheter to the third mark.This should put a roughly two to five centimeters above the iliac bifurcation. Next, inflate the balloon with 0.1 milliliters of normal saline. Now, clip the catheter and pull it back six centimeters while rotating it to damage the tissue.Once retreated, deflate the balloon. And to ensure complete endothelium dilatation, repeat the process twice more. Then remove the catheter and immediately tie off the ligature loop just above the arteriotomy site to stop the bleeding.Next, apply a suitable antiseptic around the perforate of the wound and swab away the blood clots. Lastly, close the skin incision and disinfect the surgery site with povidone-iodine solution. Then repeat the entire surgical procedure on the contralateral iliac artery using the second catheter.After the surgery, first clean the abtonic ointment from the rabbit's sides. Then administer sulfadoxine and trimethoprim. Then transfer the rabbit with the heading pad into a clean recovery cage and a monitoring patch and clips.Once the animal regains external recumbency and is ambulating, return it to its home cage. For pain management, provide buprenorphine. Now, start feeding the animal on an atherogenic diet for as long as needed.To harvest early thin flax, two or four weeks post surgery, first anesthetize the rabbit and then euthanize the rabbit with an intercardinal incision. Next, open the abdomen and expose the retroperitoneum. Then trace the aorta towards the iliac bifurcation and tie it above the bifurcation.Follow by carefully removing the surrounding tissues and isolating both iliac arteries. Now dissect out both arteries and immerse them in ice cold PBS. Therein remove the clots using forceps and divide each artery into 46 segments.Then immediately embed the segments in a mold containing OCT compound and snap freeze tissues in liquid nitrogen. Store the tissues at minus 80 degree Celsius and later stain five micron thick sections using standard methodology. Balloon injury of the iliac arteries was performed successfully without complication in under an hour.Rabbit's recovered within one hour and/or appeared healthy without significant weight loss. No post operative complications were encountered. And after four weeks of atherogenic diet, the rabbits all had hypercholesterolemia.The combination of a balloon injury and the cholesterol diet resulted in structural changes in the vessel wall leading to the development of atheroschlorotic plax in two weeks. Uninjured and balloon injured iliac arteries were isolated from the same animal and compared. Flax were characterized by lipid infiltration as well as smooth muscle sound migration and proliferation.The flax continue to grow in the injured vessels while the lumen size continue to shrink. The resulting changes were very pronounced by four weeks. After two and four weeks, morphometric quantification revealed an increased intima media thickness, an increased plaque area, a decreased lumen area, and finally an increased in lipid accumulation.Staining with alpha smooth muscle actin anti body reacts with muscle actin, thus identifying vascular smooth muscle cells. While staining with more 11 monochlonal antibody reveals the cytoplasm of macrophages. Together the stains reveal the extent to which both smooth muscle cells and macrophage proliferate into the atherosclerotic plaque in the injured vessels.After watching this video, you should have a good understanding of auto safety and easily perform a violent injury on the rabbit's iliac arteries. This injury with atherogenic diet results in plaque's developing of the iliac arteries in homogenous on time fashion.

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Research

JoVE Journal

Medicine

A Murine Model of Myocardial Ischemia-reperfusion Injury through Ligation of the Left Anterior Descending Artery

Acute or chronic myocardial infarction (MI) are cardiovascular events resulting in high morbidity and mortality. Establishing the pathological mechanisms at work during MI and developing effective therapeutic approaches requires methodology to reproducibly simulate the clinical incidence and reflect the pathophysiological changes associated with MI. Here, we describe a surgical method to induce MI in mouse models that can be used for short-term ischemia-reperfusion (I/R) injury as well as permanent ligation. The major advantage of this method is to facilitate location of the left anterior descending artery (LAD) to allow for accurate ligation of this artery to induce ischemia in the left ventricle of the mouse heart. Accurate positioning of the ligature on the LAD increases reproducibility of infarct size and thus produces more reliable results. Greater precision in placement of the ligature will improve the standard surgical approaches to simulate MI in mice, thus reducing the number of experimental animals necessary for statistically relevant studies and improving our understanding of the mechanisms producing cardiac dysfunction following MI. This mouse model of MI is also useful for the preclinical testing of treatments targeting myocardial damage following MI.

We introduce a surgical method to induce experimental ischemia/reperfusion (I/R) injury to simulate myocardial infarction (MI) in mouse models that allows for more clarity in positioning of the ligation on the left anterior descending artery (LAD) to increase the reproducibility of MI experiments in mice.

Acute or chronic myocardial infarction (MI) are cardiovascular events resulting in high morbidity and mortality. Establishing the pathological mechanisms ...

Vascular Balloon Injury and Intraluminal Administration in Rat Carotid Artery

The carotid artery balloon injury model in rats has been well established for over two decades. It remains an important method to study the molecular and cellular mechanisms involved in vascular smooth muscle dedifferentiation, neointima formation and vascular remodeling. Male Sprague-Dawley rats are the most frequently employed animals for this model. Female rats are not preferred as female hormones are protective against vascular diseases and thus introduce a variation into this procedure. The left carotid is typically injured with the right carotid serving as a negative control. Left carotid injury is caused by the inflated balloon that denudes the endothelium and distends the vessel wall. Following injury, potential therapeutic strategies such as the use of pharmacological compounds and either gene or shRNA transfer can be evaluated. Typically for gene or shRNA transfer, the injured section of the vessel lumen is locally transduced for 30 min with viral particles encoding either a protein or shRNA for delivery and expression in the injured vessel wall. Neointimal thickening representing proliferative vascular smooth muscle cells usually peaks at 2 weeks after injury. Vessels are mostly harvested at this time point for cellular and molecular analysis of cell signaling pathways as well as gene and protein expression. Vessels can also be harvested at earlier time points to determine the onset of expression and/or activation of a specific protein or pathway, depending on the experimental aims intended. Vessels can be characterized and evaluated using histological staining, immunohistochemistry, protein/mRNA assays, and activity assays. The intact right carotid artery from the same animal is an ideal internal control. Injury-induced changes in molecular and cellular parameters can be evaluated by comparing the injured artery to the internal right control artery. Likewise, therapeutic modalities can be evaluated by comparing the injured and treated artery to the control injured only artery.

This protocol uses a balloon catheter to cause an intraluminal injury on the rat carotid artery and henceforth elicit neointimal hyperplasia. This is a well-established model for studying the mechanisms of vascular remodeling in response to injury. It is also widely used to determine the validity of potential therapeutic approaches.

The carotid artery balloon injury model in rats has been well established for over two decades. It remains an important method to study the molecular and ...

A Rat Carotid Balloon Injury Model to Test Anti-vascular Remodeling Therapeutics

The rat carotid balloon injury is a well-established surgical model that has been used to study arterial remodeling and vascular cell proliferation. It is also a valuable model system to test, and to evaluate therapeutics and drugs that negate maladaptive remodeling in the vessel. The injury, or barotrauma, in the vessel lumen caused by an inflated balloon via an inserted catheter induces subsequent neointimal growth, often leading to hyperplasia or thickening of the vessel wall that narrows, or obstructs the lumen. The method described here is sufficiently sensitive, and the results can be obtained in relatively short time (2 weeks after the surgery). The efficacy of the drug or therapeutic against the induced-remodeling can be evaluated either by the post-mortem pathological and histomorphological analysis, or by ultrasound sonography in live animals. In addition, this model system has also been used to determine the therapeutic window or the time course of the administered drug. These studies can leadto the development of a better administrative strategy and a better therapeutic outcome. The procedure described here provides a tool for translational studies that bring drug and therapeutic candidates from bench research to clinical applications.

The rat carotid balloon injury model described below allows researchers to evaluate drugs or therapeutics that negate injury-induced arterial hyperplasia. Detailed pre-surgical preparation, surgical procedure, and post-surgical cares of the animal are described.

The rat carotid balloon injury is a well-established surgical model that has been used to study arterial remodeling and vascular cell proliferation. It is ...

Induction of Accelerated Atherosclerosis in Mice: The "Wire-Injury" Model

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. Moreover, by rupture of the defective vascular wall, atherosclerosis induces occlusive thrombus formation, which represents the main cause of myocardial infarction or stroke and the most frequent cause of death. Despite the advances in the cardiovascular field, many questions remain unanswered, and additional basic research is essential to improve our understanding of the molecular mechanisms during atherosclerosis and its effects. Due to limited clinical studies, there is a need for representative animal models recreating atherosclerotic conditions such as neointima formation after stent implantation, balloon angioplasty, or endarterectomy. Since the mouse presents many advantages and is the most frequently used model for studying molecular processes, the current study proposes an invasive procedure of endothelial denudation, also known as the wire-injury model, which is representative of the human condition of neointima formation in arteries after revascularization procedures.

Induction of Accelerated Atherosclerosis in Mice: The &quot;Wire-Injury&quot; Model

This study describes an invasive procedure for the induction of accelerated atherosclerosis in mice. In comparison to other methods using electric- or cryo-induced injury, mechanical-induced injury mimics the human condition of restenosis after revascularization therapies and is ideal for the study of the molecular mechanisms involved.

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. ...

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 ...

In Vivo Gene Transfer to the Rabbit Common Carotid Artery Endothelium

The goal of this method is to introduce a transgene into the endothelium of isolated segments of both rabbit common carotid arteries. The method achieves focal endothelial-selective transgenesis, thereby allowing an investigator to determine the biological roles of endothelial-expressed transgenes and to quantify the in vivo transcriptional activity of DNA sequences in large artery endothelial cells. The method uses surgical isolation of rabbit common carotid arteries and an arteriotomy to deliver a transgene-expressing viral vector into the arterial lumen. A short incubation period of the vector in the lumen, with subsequent aspiration of the lumen contents, is sufficient to achieve efficient and durable expression of the transgene in the endothelium, with no detectable transduction or expression outside of the isolated arterial segment. The method allows assessment of the biological activities of transgene products both in normal arteries and in models of human vascular disease, while avoiding systemic effects that could be caused either by targeting gene delivery to other sites (e.g. the liver) or by the alternative approach of delivering genetic constructs to the endothelium by germ line transgenesis. Application of the method is limited by the need for a skilled surgeon and anesthetist, a well-equipped operating room, the costs of purchasing and housing rabbits, and the need for expertise in gene-transfer vector construction and use. Results obtained with this method include: transgene-related alterations in arterial structure, cellularity, extracellular matrix, or vasomotor function; increases or reductions in arterial inflammation; alterations in vascular cell apoptosis; and progression, retardation, or regression of diseases such as intimal hyperplasia or atherosclerosis. The method also allows measurement of the ability of native and synthetic DNA regulatory sequences to alter transgene expression in endothelial cells, providing results that include: levels of transgene mRNA, levels of transgene protein, and levels of transgene enzymatic activity.

In Vivo Gene Transfer to the Rabbit Common Carotid Artery Endothelium

This method is to introduce a transgene into the endothelium of rabbit carotid arteries. Introduction of the transgene allows the assessment of the biological role of the transgene product either in normal arteries or disease models. The method is also useful for measuring activity of DNA regulatory sequences.

The goal of this method is to introduce a transgene into the endothelium of isolated segments of both rabbit common carotid arteries. The method achieves ...

An Experimental Model of Diet-Induced Metabolic Syndrome in Rabbit: Methodological Considerations, Development, and Assessment

In recent years, obesity and metabolic syndrome (MetS) have become a growing problem for public health and clinical practice, given their increased prevalence due to the rise of sedentary lifestyles and unhealthy eating habits. Thanks to animal models, basic research can investigate the mechanisms underlying pathological processes such as MetS. Here, we describe the methods used to develop an experimental rabbit model of diet-induced MetS and its assessment. After a period of acclimation, animals are fed a high-fat (10% hydrogenated coconut oil and 5% lard), high-sucrose (15% sucrose dissolved in water) diet for 28 weeks. During this period, several experimental procedures were performed to evaluate the different components of MetS: morphological and blood pressure measurements, glucose tolerance determination, and the analysis of several plasma markers. At the end of the experimental period, animals developed central obesity, mild hypertension, pre-diabetes, and dyslipidemia with low HDL, high LDL, and an increase of triglyceride (TG) levels, thus reproducing the main components of human MetS. This chronic model allows new perspectives for understanding the underlying mechanisms in the progression of the disease, the detection of preclinical and clinical markers that allow the identification of patients at risk, or even the testing of new therapeutic approaches for the treatment of this complex pathology.

We describe methods to develop an experimental model of diet-induced metabolic syndrome (MetS) in rabbits using a high-fat, high-sucrose diet. Animals developed central obesity, mild hypertension, pre-diabetes, and dyslipidemia, thus reproducing the main components of human MetS. This chronic model will allow acquisition of knowledge underlying mechanisms of disease progression.

In recent years, obesity and metabolic syndrome (MetS) have become a growing problem for public health and clinical practice, given their increased ...

A Rabbit Model of Durable Transgene Expression in Jugular Vein to Common Carotid Artery Interposition Grafts

Vein graft bypass surgery is a common treatment for occlusive arterial disease; however, long-term success is limited by graft failure due to thrombosis, intimal hyperplasia, and atherosclerosis. The goal of this article is to demonstrate a method for placing bilateral venous interposition grafts in a rabbit, then transducing the grafts with a gene transfer vector that achieves durable transgene expression. The method allows the investigation of the biological roles of genes and their protein products in normal vein graft homeostasis. It also allows the testing of transgenes for the activities that could prevent vein graft failure, e.g., whether the expression of a transgene prevents the neointimal growth, reduces the vascular inflammation, or reduces atherosclerosis in rabbits fed with a high-fat diet. During an initial survival surgery, the segments of right and left external jugular vein are excised and placed bilaterally as reversed end-to-side common carotid artery interposition grafts. During a second survival surgery, performed 28 days later, each of the grafts is isolated from the circulation with vascular clips and the lumens are filled (via an arteriotomy) with a solution containing a helper-dependent adenoviral (HDAd) vector. After a 20-min incubation, the vector solution is aspirated, the arteriotomy is repaired, and flow is restored. The veins are harvested at time points dictated by individual experimental protocols. The 28-day delay between the graft placement and the transduction is necessary to ensure the adaptation of the vein graft to the arterial circulation. This adaptation avoids rapid loss of transgene expression that occurs in vein grafts transduced before or immediately after grafting. The method is unique in its ability to achieve durable, stable transgene expression in grafted veins. Compared to other large animal vein graft models, rabbits have advantages of low cost and easy handling. Compared to rodent vein graft models, rabbits have larger and easier-to-manipulate blood vessels that provide abundant tissue for analysis.

This method describes the placement of interposition vein grafts in rabbits, the transduction of the grafts, and the achievement of durable transgene expression. This allows the investigation of physiological and pathological roles of transgenes and their protein products in grafted veins, and testing of gene therapies for vein graft disease.

Vein graft bypass surgery is a common treatment for occlusive arterial disease; however, long-term success is limited by graft failure due to thrombosis, ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

A Rat Carotid Artery Pressure-Controlled Segmental Balloon Injury with Periadventitial Therapeutic Application

Cardiovascular disease remains the leading cause of death and disability worldwide, in part due to atherosclerosis. Atherosclerotic plaque narrows the luminal surface area in arteries thereby reducing adequate blood flow to organs and distal tissues. Clinically, revascularization procedures such as balloon angioplasty with or without stent placement aim to restore blood flow. Although these procedures reestablish blood flow by reducing plaque burden, they damage the vessel wall, which initiates the arterial healing response. The prolonged healing response causes arterial restenosis, or re-narrowing, ultimately limiting the long-term success of these revascularization procedures. Therefore, preclinical animal models are integral for analyzing the pathophysiological mechanisms driving restenosis, and provide the opportunity to test novel therapeutic strategies. Murine models are cheaper and easier to operate on than large animal models. Balloon or wire injury are the two commonly accepted injury modalities used in murine models. Balloon injury models in particular mimic the clinical angioplasty procedure and cause adequate damage to the artery for the development of restenosis. Herein we describe the surgical details for performing and histologically analyzing the modified, pressure-controlled rat carotid artery balloon injury model. Additionally, this protocol highlights how local periadventitial application of therapeutics can be used to inhibit neointimal hyperplasia. Lastly, we present light sheet fluorescence microscopy as a novel approach for imaging and visualizing the arterial injury in three-dimensions.

The rat carotid artery balloon injury mimics the clinical angioplasty procedure performed to restore blood flow in atherosclerotic vessels. This model induces the arterial injury response by distending the arterial wall, and denuding the intimal layer of endothelial cells, ultimately causing remodeling and an intimal hyperplastic response.

Cardiovascular disease remains the leading cause of death and disability worldwide, in part due to atherosclerosis. Atherosclerotic plaque narrows the ...