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 were switched to atherogenic diet consisting of high fat (8.6%), and saturated fatty acids with 205 mg/kg cholesterol (1%) diet for the whole study duration. Balloon injury in the left iliac artery was performed 1 week after diet initiation and animals were sacrificed after 2 weeks or 4 weeks of balloon injury.
1. Preoperative Procedures
<ol>
	<li>Sterilize all surgical instruments before use with a glass bead sterilizer or other suitable instrument.</li>
	<li>Prepare and check the balloon catheter assembly.
	<ol>
		<li>Attach a 1 mL luer lock syringe filled with normal saline to the luer-lock part of the balloon catheter. Carefully monitor for absence of trapped air. Check the leaks and ensure proper balloon inflation by pressing the plunger of the syringe.</li>
	</ol>
	</li>
	<li>Weigh the rabbit and turn on the thermopad to 37 &#176;C.</li>
	<li>Use a buprenorphine solution at a concentration of 0.3 mg/ml. Inject a dose of 0.01 mg/kg subcutaneously.</li>
	<li>Anesthetize the rabbit with 5% isoflurane and 5 L/min O2 in an induction chamber for 10 - 15 min.</li>
	<li>Place the anesthetized rabbit on the heating pad kept on the surgical platform. Place the patch and clips to monitor the temperature, respiration, and electrocardiogram.</li>
	<li>Attach the snout of the rabbit to a face mask connected to a suitable anaesthesia machine. Maintain the anaesthesia with isoflurane (4.0% with 2.5 L/min O2). Confirm proper anesthetization (indicated by lack of muscle tone and loss of gag and pinnae reflexes).</li>
	<li>Apply ophthalmic ointment to both eyes to prevent the corneas from drying. Drape the rabbit with a sterile surgical sheet with only the lower limb exposed.</li>
</ol>
2. Surgical Protocol
<ol>
	<li>Remove the hair from the ventral area just below the knee joints using animal hair clippers.</li>
	<li>Swab the area with suitable disinfectant to clean the skin and remove loose hair.</li>
	<li>Locate the saphenous artery and make a small skin incision of about 1.5 cm in length using a scalpel.</li>
	<li>Expose a small portion of saphenous artery with small curved forceps without damaging the femoral vein and femoral nerve.</li>
	<li>Place two loose ligature loops (5-0 silk) beneath the saphenous artery and tie one ligature loop towards the distal end of the artery. Place a microvascular clamp above the ligature to stop the blood flow from the iliac artery.</li>
	<li>Topically apply one drop of papaverine to dilate the artery and to prevent vasospasm.</li>
	<li>Lift up the saphenous artery with the help of the tied ligature and make a small arteriotomy incision using a 24 gauge needle.</li>
	<li>Elevate the incision flaps with fine forceps and slowly insert a vein pick or a guiding needle into the lumen of the artery.</li>
	<li>Insert a 2 French Fogarty arterial embolectomy catheter into the saphenous artery. Remove the vein pick and microvascular clamps.</li>
	<li>Advance the catheter till the sixth mark (20 - 25 cm) corresponding to a position roughly 2-5 cm above the iliac bifurcation.</li>
	<li>Inflate the balloon with 0.1 mL normal saline using a 1 mL syringe or at a nominal pressure of 6 atm using a regulated manual inflator as described in 16,22.</li>
	<li>Hold the balloon catheter with forceps and drag back by 6 cm through the iliac arterytoward the point of insertion, while rotating the catheter.</li>
	<li>Deflate the balloon by pulling back the plunger of the syringe.</li>
	<li>Repeat steps 2.10 to 2.13 three times to ensure complete endothelial denudation.</li>
	<li>Remove the catheter and immediately tie the ligature loop just above the arteriotomy site to stop bleeding.</li>
	<li>Apply suitable antiseptic all around the periphery of the wound and swab away the blood clots. Close the skin incision with a 5-0 suture, and disinfect the surgery site with povidone-iodine solution.</li>
	<li>Repeat steps 2.1 to 2.16 on the contralateral iliac using a new catheter.</li>
	<li>Swab the ophthalmic ointment from eyes.</li>
</ol>
3. Post-operative Care
<ol>
	<li>Administer sulfadoxine 40 mg/kg and trimethoprim 8 mg/kg or any other suitable antibiotic immediately after the surgical procedure.</li>
	<li>During the anesthesia-recovery period, keep the rabbit over a heat pad placed in a clean autoclaved cage.</li>
	<li>Remove the monitoring patch and clips.</li>
	<li>After recovery, return the rabbits to their home cages. Inject subcutaneously buprenorphine 0.05 - 0.1 mg/kg post -operatively every 6 - 12 h for 48 h. Continue atherogenic diet for another two weeks or four weeks.</li>
</ol>
4. Tissue Harvesting and Analysis of Plaque Composition
<ol>
	<li>After two weeks (for early thin plaque) or three weeks of balloon injury, anesthetize the rabbit using isoflurane in a similar way as described above.</li>
	<li>Open the thoracic cavity and euthanize the rabbits by intracardial exsanguination.</li>
	<li>Isolate the iliac arteries as described in 23.
	<ol>
		<li>Briefly, open the abdomen and expose the retroperitoneum. Trace the aorta towards the iliac bifurcation and tie it above the bifurcation. Carefully remove the surrounding tissues to expose and isolate both iliac arteries.</li>
	</ol>
	</li>
	<li>Dissect out both iliac arteries and immerse them in ice-cold phosphate buffered saline. Remove the clots with the help of forceps. Divide each iliac artery into 4 - 6 segments to characterise the thickness of the plaque throughout the artery.</li>
	<li>Immediately embed the arterial segments in a mold containing optimum cutting temperature compound, snap-freeze using liquid nitrogen and keep it at -70 &#176;C. Prepare 5 &#181;m thick sections using a cryostat as described in 24.</li>
	<li>Perform histology, immunofluorescence or immunohistochemical staining for morphometry, plaque lipid and cellular content as described in 10,25. 
	NOTE: Briefly, rinse the arterial sections with phosphate buffered saline (PBS) and permeabilize using 0.2 % Triton. Rinse the sections with PBS and block non specific sites with 2% bovine serum albumin for 30 min. Incubate the sections for 1 h at 37 &#176;C with anti &#945;-SM actin (1:200) or RAM11 antibody (1:200). Rinse the sections with PBS and incubate them with appropriate secondary antibody for 30 min at 37 &#176;C. Wash again with PBS and add Hoechst (5 &#181;g/mL) for 10 min to detect nuclei.</li>
</ol>

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The authors declare no competing financial interests.

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><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&amp;auml;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&amp;agrave;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&#039;361</td><td></td></tr><tr><td>Antiseptic- Octenisept</td><td>Sch&amp;uuml;lke &amp;amp; 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&#039;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>&amp;alpha;-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-rabbit-model-accelerated-atherosclerosis-methodological

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

12.0K Views.  University of Fribourg. 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. 

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

A Model of Disturbed Flow-Induced Atherosclerosis in Mouse Carotid Artery by Partial Ligation and a Simple Method of RNA Isolation from Carotid Endothelium

Despite the well-known close association, direct evidence linking disturbed flow to atherogenesis has been lacking. We have recently used a modified version of carotid partial ligation methods [1,2] to show that it acutely induces low and oscillatory flow conditions, two key characteristics of disturbed flow, in the mouse common carotid artery. Using this model, we have provided direct evidence that disturbed flow indeed leads to rapid and robust atherosclerosis development in Apolipoprotein E knockout mouse [3]. We also developed a method of endothelial RNA preparation with high purity from the mouse carotid intima [3]. Using this mouse model and method, we found that partial ligation causes endothelial dysfunction in a week, followed by robust and rapid atheroma formation in two weeks in a hyperlipidemic mouse model along with features of complex lesion formation such as intraplaque neovascularization by four weeks. This rapid in vivo model and the endothelial RNA preparation method could be used to determine molecular mechanisms underlying flow-dependent regulation of vascular biology and diseases. Also, it could be used to test various therapeutic interventions targeting endothelial dysfunction and atherosclerosis in considerably reduced study duration.

This describes a partial carotid ligation surgery, which causes disturbed flow conditions and subsequent atherosclerosis development (in two weeks) with intraplaque neo-vascularization (in four weeks) in the mouse common carotid artery. We also describe a novel method of RNA isolation from the carotid intima, providing high purity endothelial RNA.

Despite the well-known close association, direct evidence linking disturbed flow to atherogenesis has been lacking. We have recently used a modified ...

In vivo Near Infrared Fluorescence (NIRF) Intravascular Molecular Imaging of Inflammatory Plaque, a Multimodal Approach to Imaging of Atherosclerosis

The vascular response to injury is a well-orchestrated inflammatory response triggered by the accumulation of macrophages within the vessel wall leading to an accumulation of lipid-laden intra-luminal plaque, smooth muscle cell proliferation and progressive narrowing of the vessel lumen. The formation of such vulnerable plaques prone to rupture underlies the majority of cases of acute myocardial infarction. The complex molecular and cellular inflammatory cascade is orchestrated by the recruitment of T lymphocytes and macrophages and their paracrine effects on endothelial and smooth muscle cells.1
Molecular imaging in atherosclerosis has evolved into an important clinical and research tool that allows in vivo visualization of inflammation and other biological processes. Several recent examples demonstrate the ability to detect high-risk plaques in patients, and assess the effects of pharmacotherapeutics in atherosclerosis.4 While a number of molecular imaging approaches (in particular MRI and PET) can image biological aspects of large vessels such as the carotid arteries, scant options exist for imaging of coronary arteries.2 The advent of high-resolution optical imaging strategies, in particular near-infrared fluorescence (NIRF), coupled with activatable fluorescent probes, have enhanced sensitivity and led to the development of new intravascular strategies to improve biological imaging of human coronary atherosclerosis.
Near infrared fluorescence (NIRF) molecular imaging utilizes excitation light with a defined band width (650-900 nm) as a source of photons that, when delivered to an optical contrast agent or fluorescent probe, emits fluorescence in the NIR window that can be detected using an appropriate emission filter and a high sensitivity charge-coupled camera. As opposed to visible light, NIR light penetrates deeply into tissue, is markedly less attenuated by endogenous photon absorbers such as hemoglobin, lipid and water, and enables high target-to-background ratios due to reduced autofluorescence in the NIR window. Imaging within the NIR 'window' can substantially improve the potential for in vivo imaging.2,5
Inflammatory cysteine proteases have been well studied using activatable NIRF probes10, and play important roles in atherogenesis. Via degradation of the extracellular matrix, cysteine proteases contribute importantly to the progression and complications of atherosclerosis8. In particular, the cysteine protease, cathepsin B, is highly expressed and colocalizes with macrophages in experimental murine, rabbit, and human atheromata.3,6,7 In addition, cathepsin B activity in plaques can be sensed in vivo utilizing a previously described 1-D intravascular near-infrared fluorescence technology6, in conjunction with an injectable nanosensor agent that consists of a poly-lysine polymer backbone derivatized with multiple NIR fluorochromes (VM110/Prosense750, ex/em 750/780nm, VisEn Medical, Woburn, MA) that results in strong intramolecular quenching at baseline.10 Following targeted enzymatic cleavage by cysteine proteases such as cathepsin B (known to colocalize with plaque macrophages), the fluorochromes separate, resulting in substantial amplification of the NIRF signal. Intravascular detection of NIR fluorescence signal by the utilized novel 2D intravascular NIRF catheter now enables high-resolution, geometrically accurate in vivo detection of cathepsin B activity in inflamed plaque.
In vivo molecular imaging of atherosclerosis using catheter-based 2D NIRF imaging, as opposed to a prior 1-D spectroscopic approach,6 is a novel and promising tool that utilizes augmented protease activity in macrophage-rich plaque to detect vascular inflammation.11,12 The following research protocol describes the use of an intravascular 2-dimensional NIRF catheter to image and characterize plaque structure utilizing key aspects of plaque biology. It is a translatable platform that when integrated with existing clinical imaging technologies including angiography and intravascular ultrasound (IVUS), offers a unique and novel integrated multimodal molecular imaging technique that distinguishes inflammatory atheromata, and allows detection of intravascular NIRF signals in human-sized coronary arteries.

In vivo Near Infrared Fluorescence NIRF Intravascular Molecular Imaging of Inflammatory Plaque, a Multimodal Approach to Imaging of Atherosclerosis

We detail a new near-infrared fluorescence (NIRF) catheter for 2-dimensional intravascular molecular imaging of plaque biology in vivo. The NIRF catheter can visualize key biological processes such as inflammation by reporting on the presence of plaque-avid activatable and targeted NIR fluorochromes. The catheter utilizes clinical engineering and power requirements and is targeted for application in human coronary arteries. The following research study describes a multimodal imaging strategy that utilizes a novel in vivo intravascular NIRF catheter to image and quantify inflammatory plaque in proteolytically active inflamed rabbit atheromata.

The vascular response to injury is a well-orchestrated inflammatory response triggered by the accumulation of macrophages within the vessel wall leading ...

Inducing Myointimal Hyperplasia Versus Atherosclerosis in Mice: An Introduction of Two Valid Models

Various in vivo laboratory rodent models for the induction of artery stenosis have been established to mimic diseases that include arterial plaque formation and stenosis, as observed for example in ischemic heart disease. Two highly reproducible mouse models&#160;&#8211;&#160;both resulting in artery stenosis but each underlying a different pathway of development&#160;&#8211; are introduced here. The models represent the two most common causes of artery stenosis; namely one mouse model for each myointimal hyperplasia, and atherosclerosis are shown. To induce myointimal hyperplasia, a balloon catheter injury of the abdominal aorta is performed. For the development of atherosclerotic plaque, the ApoE -/- mouse model in combination with western fatty diet is used. Different model-adapted options for the measurement and evaluation of the results are named and described in this manuscript. The introduction and comparison of these two models provides information for scientists to choose the appropriate artery stenosis model in accordance to the scientific question asked.

This video shows two models of intimal plaque development in murine arteries and emphasizes the differences in myointimal hyperplasia and atherosclerosis.

Various in vivo laboratory rodent models for the induction of artery stenosis have been established to mimic diseases that include arterial plaque ...

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

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

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

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

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.

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. ...

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.

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 ...

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 ...

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 ...