The authors thank Christiane Pahrmann for her technical assistance.
D.W. was supported by the Max Kade Foundation. T.D. received grants from the Else Kro&#776;ner Fondation (2012_EKES.04) and the Deutsche Forschungsgemeinschaft (DE2133/2-1_. S. S. received research grants from the Deutsche Forschungsgemeinschaft (DFG; SCHR992/3- 1, SCHR992/4-1).

Animals received humane care in compliance with the Guide for the Principles of Laboratory Animals, prepared by the Institute of Laboratory Animal Resources, and published by the National Institutes of Health. All animal protocols were approved by the responsible local authority (&#39;&#39;Amt f&#252;r Gesundheit und Verbraucherschutz, Hansestadt (Office for Health and Consumer Protection) Hamburg&#39;&#39;).
1. Catheter Preparation
NOTE: Refer to the Table of Materials for information regarding the catheter.
<ol>
	<li>Take catheter from the catheter holder.</li>
	<li>Pull the guidewire from the lumen through the distal port out.</li>
	<li>Put in a drop of cyanoacrylate adhesive on the distal end of catheter. 
	Caution: Wear latex or nitrile gloves.</li>
	<li>Place the guidewire at the RX port of the catheter and advance it through the lumen to the distal port. Afterwards, pull the guidewire back slightly to leave a space of ~5 mm to the end.</li>
	<li>Wait 5 min to allow the adhesive to dry.</li>
	<li>Move the guidewire, it should be fixed. If it is still mobile, repeat steps 1.3 - 1.6.</li>
	<li>Fill a 3-mL syringe with 1.5 mL 0.9% saline and connect it to the balloon inflation port.</li>
	<li>Push the syringe plunger to test balloon inflation. Leave 0.6 mL saline in the syringe.</li>
</ol>
2. Mouse preparation
<ol>
	<li>Obtain male mice at the age of 14 weeks weighing approximately 30 g. 
	NOTE: We used animals obtained from the Institute of Laboratory Animals. We used C57BL/6J here.</li>
	<li>Use an induction chamber to anaesthetize the mouse with 2.0-2.5% isofurane (500 mL/min oxygen flow rate).</li>
	<li>Place the mouse on its back on a heating pad and maintain anesthesia with a facemask covering the mouth and nose of the mouse. Check for sufficient depth of anesthesia by pinching the hind feet and tail to verify an absence of reflexes.</li>
	<li>Remove the abdominal hair using a hair trimmer.</li>
	<li>Spread the hind legs and fix their position using tape.</li>
	<li>Disinfect the abdominal area using povidone-iodine, followed by 80% ethanol. Repeat this step two more times.</li>
	<li>Use a surgical drape to ensure surgical site does not get contaminated.&#160;Open the skin and muscle layers along the linea alba with a scissor (or scalpel) to expose the abdominal organs.</li>
	<li>Lay the intestines in a 0.9% saline moisturized glove and wrap to keep them moist.</li>
	<li>Use two fine forceps to remove the fatty tissue above the abdominal aorta.</li>
	<li>Using an insulin syringe (30G), inject 250 &#181;L of heparin solution (50 U/mL) into the Inferior Vena Cava (IVC) and wait 3 min for its systemic distribution.&#160;Heparin will suppress hemostasis and prevent undesired clotting during surgery.</li>
	<li>Using two forceps, dissect the infrarenal aorta down to its bifurcation and its outgoing branch vessels.</li>
	<li>Ligate the side vessels, which are expected to be placed in the clamped area, with a high temperature cauterizer.</li>
	<li>Stop blood flow by clamping the infrarenal aorta directly beneath the renal arteries.</li>
	<li>Place a second clamp at a distal position just above the aortic bifurcation.</li>
	<li>Perform a small horizontal incision using scissors at the midpoint between the clamps along the vessel. 
	NOTE: The size of the incision should be equivalent to 1/3 of the circumference of the vessel.</li>
	<li>Insert a syringe with a (30G) needle into the incision and flush the aorta with 250 &#181;L heparin solution (50 U/mL).</li>
	<li>Using 10-0 sutures, place one single knot on each side of the incision.</li>
	<li>Dilate the aorta by inserting a vessel dilator into the incision and spread the vessel slightly. Repeat the dilation 2 to 3 times.</li>
	<li>Moisturize the balloon-catheter with 0.9% saline.</li>
	<li>Insert the flattened balloon-catheter into the aorta and advance it towards the proximal clamp on the aorta.</li>
	<li>When reaching the proximal clamp, carefully open the proximal clamp and inflate the balloon to prevent blood leakage, by injecting ~0.6 mL of saline. 
	NOTE: The ratio of the inflated balloon to vessel is 1.5:1.</li>
	<li>Advance the catheter retrograde for approximately 2 cm.</li>
	<li>Pull the expanded catheter back, while deflating the balloon slightly by releasing the syringe.</li>
	<li>Reattach the proximal clamp when the catheter reaches the incision of the aorta. Deflate the balloon completely and remove it.</li>
	<li>Rinse the aorta with 250 &#181;L heparin solution (50 U/mL) using a 30G syringe.</li>
	<li>Close the aortic incision using 10-0 sutures. Place interrupted stitches on each lateral side, followed by one or two stitches on the ventral side.</li>
	<li>Open the distal clamp. In case of bleeding, close the clamp again and place additional stitches.</li>
	<li>Open the proximal clamp carefully.</li>
	<li>Place two swabs on the suture to support it and stop any bleeding.</li>
	<li>Place absorbable hemostats on the suture to sustain it. 
	NOTE: An aortic pulse should be visible distally from the incision.</li>
	<li>Place the intestines back into the abdomen.</li>
	<li>Rinse the abdominal cavity with sterile 0.9% saline, which has been pre-warmed to 37 &#176;C.</li>
	<li>Close the abdominal muscle layer using 6-0 running sutures.</li>
	<li>Close the skin with 5-0 running sutures.</li>
	<li>Inject 4-5 mg/kg Carprofen subcutaneously before allowing the mouse to wake up. Monitor the animal until it has gained consciousness, maintain sternal recumbency. Keep the animal alone in a cage until complete recovery.</li>
	<li>Add Metamizole to the drinking water (50 mg/100 mL) as pain medication for 3 days and monitor the animal daily.&#160;Usually mice like the sweet taste of metamizole and start drinking immediately after the surgery. If preferred, sustained-release injectable agents can be used instead of metamizole. 
	NOTE: The observation period for this model is 28 days.</li>
</ol>
3. Histopathology
<ol>
	<li>Harvest the balloon-injured aorta after 28 days by preparing the mice as described in steps 2.2 to 2.9.</li>
	<li>Use scissors to remove the balloon-injured aorta (between the bifurcation and 0.3 mm above the renal vessels) and euthanize the mouse by cutting out its heart.</li>
	<li>Flush the lumen of the vessel with 0.9% NaCl.</li>
	<li>Fix the harvested vessel in 4% paraformaldehyde (PFA) overnight and dehydrate it in increasing concentrations of ethanol, starting with 70% ethanol for 2 hours, 80% ethanol for 1 hour, 95% ethanol for 2 hours, and 100% ethanol for 5 hours. Then, incubate the samples in xylene for 2 hours 3 times, before infiltrating the samples with paraffin. 
	NOTE: Instead of flushing the harvested vessel with 0.9% NaCl, it can be flushed with 4% PFA. 
	Caution: PFA and xylene are toxic and should be handled with special care.</li>
	<li>Embed the sample in paraffin and cut into slices of 5 &#181;m thickness using a microtome.</li>
	<li>Deparaffinize the slides with xylene 3 times for 5 minutes.</li>
	<li>Rehydrate tissue slides using a decreasing series of ethanol. Start with 100% ethanol 2 times for 5 min, followed by 3 minutes of 95%, 80% and 70% ethanol.</li>
	<li>Stain the slides with Masson&#39;s trichrome staining as described18.</li>
	<li>Dehydrate stained slides in 100% ethanol 2 times for 10 min each. Clear with xylene 2 times for 10 min each and mount in mounting medium.</li>
	<li>View the slides with a bright field microscope. Use a lens with 5x magnification and a numerical aperture of 0.12 for an overview picture or a lens with 20x magnifications with a numerical aperture of 0.35 for detailed observation.</li>
</ol>
4. Immunofluorescence Microscopy
<ol>
	<li>Rehydrate tissue slides using a decreasing series of ethanol. Start with 100% ethanol 2 times for 5 min, followed by 3 min of 95%, 80% and 70% ethanol.</li>
	<li>Perform antigen-retrieval by heating the slides in antigen-retrieval solution in a steamer for 20 min.</li>
	<li>Let the slides cool down to room temperature.</li>
	<li>After washing the slides for three times with phosphate buffered saline (PBS), apply antigen blocking solution on sections for 30 min.</li>
	<li>Wash slides three times for 5 min with PBS.</li>
	<li>Incubate sections with primary antibody diluted in primary antibody diluent. 
	NOTE: The right concentration and incubation time should be chosen separately for each antibody.</li>
	<li>Wash slides three times for 5 min with PBS to remove unbound antibody.</li>
	<li>Incubate sections with a pre-conjugated secondary antibody diluted in secondary antibody diluent. 
	NOTE: The right concentration and incubation time should be chosen separately for each antibody.</li>
	<li>Remove unbound antibodies by washing the slides for 5 min three times.</li>
	<li>Counterstain the cell nuclei using 4&#39;,6-diamidino-2-phenylindole (DAPI) for 15 min; the final DAPI concentration should be 350 nM.</li>
	<li>Mount slides in immunofluorescence compatible mounting solution. 
	NOTE: Using the wrong mounting solution can obscure the fluorescence signal.</li>
	<li>View slides with a fluorescence microscope. Use a 40x magnification lens with a numerical aperture of 1.3.</li>
</ol>

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K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+differentiation+of+vascular+smooth+muscle+cells.">Regulation of differentiation of vascular smooth muscle cells.</a> Physiol Rev. 75 (3), 487-517 (1995).</li><li>Owens, G. K., Kumar, M. S., Wamhoff, B. 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The authors have nothing to disclose.

This article demonstrates a murine model to study the development of myointimal hyperplasia and allows the exploration of the underlying pathological processes and the testing of new drugs or therapeutic options.
The most critical step in this protocol is the denudation of the aorta. Special care should be paid during this step as excessive denudation will lead to aneurysm formation and model failure. On the other hand, if denudation is performed insufficiently, too little myointima will devel...

Arterial stenosis in coronary and peripheral arteries has a large effect on the morbidity and mortality of patients1. One underlying pathological mechanism is myointima hyperplasia (MH), which is characterized by increased proliferation, migration, and synthesis of extracellular matrix proteins from vascular smooth muscle cells (SMC)2. SMC are located in the media layer of the vessel and migrate upon stimulation to the surface of the lumen. Stimulatory signals include growth factors, cytokines, cell-cell contact, lipids, extracellular matrix components, and mechanical shear and stretch forces3,4,5,6. Injuries of the vessel wall, pathological or iatrogenic, cause endothelial cell and smooth muscle cell damage and stimulate inflammatory reactions, and thus lead to MH7.
Different animal models are currently available to study arterial injury and myointima hyperplasia. Large animals like pigs or dogs have the advantage of sharing a similar artery and coronary anatomy with humans and are especially suitable for studies investigating angioplasty techniques, procedure, and devices8. However, pig models have the drawback of higher thrombogenicity9,10, while dogs only have a mild response to vessel injury11. In addition, all large animal models require special housing, equipment, and staff, which are connected with high costs and are not always available at an institution. Small animal models include rats and mice. Compared to rats, mice have the advantages of lower cost and the existence of a variety of knock out models. The model described in this video can be combined with ApoE-/- mice fed with a western diet to closely mimic the clinical setting of angioplasty in atherosclerotic vessels12. Previous models induced vascular injury via wire injury13, fluid desiccation14, spring15, or cuff injury16. Since the nature of the injury will greatly impact the development and constitution of MH, using a balloon catheter to induce vessel injury is the best way to mimic the clinical setting.
In this article we describe a novel method to induce MH with a balloon catheter in mice. The use of a balloon catheter (1.2 mm x 6 mm) with a RX-Port (Figure 1A) allows the scraping of the intimal layer and, at the same time, the induction of an overdistension of the vessel. Both of these factors are important triggers for the development of MH. The observation time for this model is 28 days17.

<table><tbody><tr><td>10-0 Ethilon suture</td><td>Ethicon</td><td>2814G</td><td></td></tr><tr><td>3 mL Syringe</td><td>BD Medical</td><td>309658</td><td></td></tr><tr><td>37% HCl</td><td>Sigma-Aldrich</td><td>H1758</td><td></td></tr><tr><td>5-0 prolene suture</td><td>Ethicon</td><td>EH7229H</td><td></td></tr><tr><td>6-0 prolene suture</td><td>Ethicon</td><td>8706H</td><td></td></tr><tr><td>Acid Fuchsin</td><td>Sigma-Aldrich</td><td>F8129-25G</td><td>Trichrome staining</td></tr><tr><td>Antigen retrieval solution</td><td>Dako</td><td>S1699</td><td></td></tr><tr><td>Azophloxin</td><td>Waldeck</td><td>1B-103</td><td>Trichrome staining</td></tr><tr><td>Bepanthen Eye and Nose ointment</td><td>Bayer</td><td>1578675</td><td>Eye ointment</td></tr><tr><td>Betadine Solution</td><td>Betadine Purdue Pharma</td><td>NDC:67618-152</td><td></td></tr><tr><td>C57BL/6J</td><td>Charles River</td><td>Stock number 000664</td><td></td></tr><tr><td>Clamp applicator</td><td>Fine Science Tools</td><td>18056-14</td><td>JAW DIMS: 4 x 0.75 mm
LENGTH: 13 mm</td></tr><tr><td>Collagen 3</td><td>abcam</td><td>ab7778</td><td>Antibody</td></tr><tr><td>DAPI</td><td>Thermo Fischer</td><td>D1306</td><td></td></tr><tr><td>Donkey anti-Goat IgG AF555</td><td>Invitrogen</td><td>A21432</td><td>Secondary antibody</td></tr><tr><td>Donkey anti-Rabbit IgG AF488</td><td>Invitrogen</td><td>A21206</td><td>Secondary antibody</td></tr><tr><td>Donkey anti-Rabbit IgG AF488</td><td>Invitrogen</td><td>A11055</td><td>Secondary antibody</td></tr><tr><td>Donkey anti-Rabbit IgG AF555</td><td>Invitrogen</td><td>A31572</td><td>Secondary antibody</td></tr><tr><td>Ethanol 70%</td><td>Th. Geyer</td><td>2270</td><td></td></tr><tr><td>Ethanol 96%</td><td>Th. Geyer</td><td>2295</td><td></td></tr><tr><td>Ethanol absolute</td><td>Th. Geyer</td><td>2246</td><td></td></tr><tr><td>FAP</td><td>abcam</td><td>ab28246</td><td>Antibody</td></tr><tr><td>Forceps fine</td><td>Fine Science Tools</td><td>11251-20</td><td></td></tr><tr><td>Forceps standard</td><td>Fine Science Tools</td><td>11023-10</td><td></td></tr><tr><td>Glacial Acetic Acid</td><td>Sigma-Aldrich</td><td>537020</td><td></td></tr><tr><td>Hair clipper</td><td>WAHL</td><td>8786-451A ARCO SE</td><td></td></tr><tr><td>Heparin</td><td>Rotexmedica</td><td>PZN 3862340</td><td>25.000 I.E./mL</td></tr><tr><td>High temperature cautery kit</td><td>Bovie</td><td>18010-00</td><td></td></tr><tr><td>Image-iT FX Signal Enhancer</td><td>Invitrogen</td><td>I36933</td><td>Blocking solution</td></tr><tr><td>Light Green SF</td><td>Waldeck</td><td>1B-211</td><td>Trichrome staining</td></tr><tr><td>Microsurgical clamp</td><td>Fine Science Tools</td><td>18055-04</td><td>Micro-Serrefine - 4mm</td></tr><tr><td>MINI TREK Coronary Dilatation Catheter 1.20 mm x 6 mm / Rapid-Exchange</td><td>Abbott</td><td>1012268-06U</td><td></td></tr><tr><td>Molybdatophosphoric acid hydrate</td><td>Merck</td><td>1.00532.0100</td><td>Trichrome staining</td></tr><tr><td>NaCl 0,9%</td><td>B.Braun</td><td>PZN 06063042 Art. Nr.: 3570160</td><td></td></tr><tr><td>Needle holder</td><td>Fine Science Tools</td><td>12075-14</td><td></td></tr><tr><td>Novaminsulfon</td><td>Ratiopharm</td><td>PZN 03530402</td><td>Metamizole</td></tr><tr><td>Orange G</td><td>Waldeck</td><td>1B-221</td><td>Trichrome staining</td></tr><tr><td>Paraffin</td><td>Leica biosystems</td><td>REF 39602004</td><td></td></tr><tr><td>PBS pH 7,4</td><td>Gibco</td><td>10010023</td><td></td></tr><tr><td>PFA 4%</td><td>Electron Microscopy Sciences</td><td>#157135S</td><td></td></tr><tr><td>Ponceau S solution</td><td>Serva Electrophoresis</td><td>33427</td><td>Trichrome staining</td></tr><tr><td>Primary antibody diluent</td><td>Dako</td><td>S3022</td><td></td></tr><tr><td>Prolong Gold Mounting solution</td><td>Thermo Fischer</td><td>P36930</td><td>Mounting solution for immunofluorescence stained slides</td></tr><tr><td>Replaceable Fine Tip</td><td>Bovie</td><td>H101</td><td></td></tr><tr><td>Resorcin-Fuchsin Weigert</td><td>Waldeck</td><td>2E-30</td><td>Trichrome staining</td></tr><tr><td>Rimadyl</td><td>Pfizer</td><td>400684.00.00</td><td>Carprofen</td></tr><tr><td>Scissors</td><td>Fine Science Tools</td><td>14028-10</td><td></td></tr><tr><td>Scissors Vannas-style</td><td>Fine Science Tools</td><td>15000-03</td><td></td></tr><tr><td>Secondary antibody diluent</td><td>Dako</td><td>S0809</td><td></td></tr><tr><td>Fast acting Adhesive MINIS 3x1g</td><td>UHU</td><td>45370</td><td>Cyanoacrylate</td></tr><tr><td>Slide Rack</td><td>Ted Pella</td><td>21057</td><td></td></tr><tr><td>SM22</td><td>abcam</td><td>ab10135</td><td>Antibody</td></tr><tr><td>SMA</td><td>abcam</td><td>ab21027</td><td>Antibody</td></tr><tr><td>Staining dish</td><td>Ted Pella</td><td>21075</td><td></td></tr><tr><td>Surgical microscope</td><td>Leica</td><td>M651</td><td></td></tr><tr><td>Tabotamp fibrillar</td><td>Ethicon</td><td>431962</td><td>Absorbable hemostat</td></tr><tr><td>Transpore Surgical Tape</td><td>3M</td><td>1527-1</td><td></td></tr><tr><td>U-100 Insulin syringe</td><td>BD Medical</td><td>324825</td><td></td></tr><tr><td>Vessel Dilator</td><td>Fine Science Tools</td><td>18603-14</td><td></td></tr><tr><td>Vitro-Clud</td><td>Langenbrinck</td><td>04-0001</td><td></td></tr><tr><td>Weigerts iron hematoxylin Kit</td><td>Merck</td><td>1.15973.0002</td><td>Trichrome staining</td></tr><tr><td>Xylene</td><td>Th. Geyer</td><td>3410</td><td></td></tr></tbody></table>

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.

Balloon denudation is a suitable model to study the development of MH in mice. Animals recover well from the surgery and show an excellent physical condition post-operation. We established this model in 50 mice with less than 3% death rate due to the surgical procedure. Figures 1B-C show the key surgical steps. After a skin incision along the linea alba, identify the aorta abdominalis. Place microsurgical clamps (<strong cla...

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Balloon-based Injury to Induce Myointimal Hyperplasia in the Mouse Abdominal Aorta

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 ...

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Research

JoVE Journal

Medicine

9.6K Views.  University Heart Center. Dit artikel demonstreert een lymfkliertest model voor het bestuderen van de ontwikkeling van myointimal hyperplasie (MH) na de ballon van de aorta blessure.

Video: Balloon-based Injury to Induce Myointimal Hyperplasia in the Mouse Abdominal Aorta

Creation of Murine Experimental Abdominal Aortic Aneurysms with Elastase 

Transient intraluminal infusion of porcine pancreatic elastase into the infrarenal segment of the abdominal aorta is the most widely used animal model of abdominal aortic aneurysm (AAA) ever since it was first described in rats by Anidjar and colleagues.1 The rationale for its development was based on the disrupted nature of elastin observed in AAAs. This rat model has been modified to produce AAAs in the infrarenal aortic region of mice.2 The model has the ability to add broad insight into the pathobiology of AAA due to the emergence of numerous transgenic and gene knockout mice. Moreover, it is a viable platform to test potential therapeutic agents for AAA. In this video, we demonstrate the elastase infusion AAA procedure used in our laboratory. 
Mice are anesthetized using 2.5% isoflurane, and a laparotomy is performed under sterile conditions. The abdominal aortais isolated with the assistance of an operating stereomicroscope (Leica). After placing temporary ligatures around the proximal and distal aorta, an aortotomy is created at the bifurcation with the tip of a 30-gauge needle. A heat-tapered segment of PE-10 polyethylene tubing is introduced through the aortotomy and secured. The aortic lumen is subsequently perfused for 5-15 minutes at 100 mm Hg with saline containing type I porcine pancreatic elastase (4.5 U/mL; Sigma Chemical Co.). After removing the perfusion catheter, the aortotomy is repaired without constriction of the lumen.

Creation of Murine Experimental Abdominal Aortic Aneurysms with Elastase

This video shows how to induce abdominal aortic aneurysms (AAA) in mice via transient intraluminal infusion of porcine pancreatic elastase into the infrarenal segment of the abdominal aorta. The model has the ability to add broad insight into the pathobiology of AAA due to the emergence of numerous transgenic and gene knockout mice.

Transient intraluminal infusion of porcine pancreatic elastase into the infrarenal segment of the abdominal aorta is the most widely used animal model of ...

Biology

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 ...

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. ...

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 ...

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.

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 ...

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 ...

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 ...

Investigating the Molecular Mechanisms Underlying Murine Abdominal Aortic Aneurysm

A Mouse Abdominal Aortic Aneurysm Model by Periadventitial Calcium Chloride and Elastase Infiltration

Abdominal aortic aneurysm (AAA) is a life-threatening disease associated with high mortality rates. It is characterized by the permanent dilation of the abdominal aorta with at least a 50% increase in arterial diameter. Various animal models of AAA have been introduced to mimic the pathophysiological changes and study the underlying mechanisms of AAA. Among these models, the calcium chloride (CaCl2)- and elastase-induced AAA models are commonly used in mice. However, these methods have certain limitations. Traditional intraluminal porcine pancreatic elastase (PPE) perfusion is associated with high technical difficulty and a high rupture rate, while periadventitial administration of PPE yields inconsistent results. In addition, the CaCl2-induced AAA model lacks human AAA features, such as atherothrombosis and aneurysm rupture. Therefore, the combined application of CaCl2 and PPE has been proposed as an approach to enhance success rates and induce greater diameter increases in AAA animal models. This manuscript presents a comprehensive protocol for establishing a mouse AAA model through periaortic infiltration of PPE and CaCl2 in the infrarenal segment of the abdominal aorta. By following this protocol, we can achieve an AAA formation rate of approximately 90% with technical simplicity and reproducibility. Further ultrasound and histological experiments confirm that this model effectively replicates the morphological and pathological changes observed in human AAA.

Author Spotlight: Development and Characterization of a Mouse Model for Abdominal Aortic Aneurysm

This manuscript presents a protocol for establishing a mouse abdominal aortic aneurysm model using calcium chloride and elastase, combining the advantages of previous modeling methods. This model can be utilized to investigate the pathophysiological mechanisms underlying abdominal aortic aneurysms.

Abdominal aortic aneurysm (AAA) is a life-threatening disease associated with high mortality rates. It is characterized by the permanent dilation of the ...

Mouse Abdominal Aortic Aneurysm Model Induced by Perivascular Application of Elastase

Abdominal aortic aneurysm (AAA), although primarily asymptomatic, is potentially life-threatening as the rupture of AAA usually has a devastating outcome. Currently, there are several distinct experimental models of AAA, each emphasizing a different aspect in the pathogenesis of AAA. The elastase-induced AAA model is the second most used rodent AAA model. This model involves direct infusion or application of porcine pancreatic elastase (PPE) to the infrarenal segment of the aorta. Due to technical challenges, most elastase-induced AAA model nowadays is performed with the external application rather than an intraluminal infusion of PPE. The infiltration of elastase will cause degradation of elastic lamellae in the medial layers, resulting in the loss of aortic wall integrity and subsequent dilation of the abdominal aorta. However, one disadvantage of the elastase-induced AAA model is the inevitable variation of how the surgery is performed. Specifically, the surgical technique of isolating the infrarenal segment of the aorta, the material used for aorta wrapping and PPE incubation, the enzymatic activity of PPE, and the time duration of PPE application can all be important determinants that affect the eventual AAA formation rate and aneurysm diameter. Notably, the difference in these factors from different studies on AAA can lead to reproducibility issues. This article describes a detailed surgical process of the elastase-induced AAA model through direct application of PPE to the adventitia of the infrarenal abdominal aorta in the mouse. Following this procedure, a stable AAA formation rate of around 80% in male and female mice is achievable. The consistency and reproducibility of AAA studies using an elastase-induced AAA model can be significantly enhanced by establishing a standard surgical procedure.

The present protocol describes a standardized surgical method for the elastase-induced AAA model through the direct application of elastase to the adventitia of infrarenal abdominal aorta in mice.

Abdominal aortic aneurysm (AAA), although primarily asymptomatic, is potentially life-threatening as the rupture of AAA usually has a devastating outcome. ...

Balloon-based Injury to Induce Myointimal Hyperplasia in the Mouse Abdominal Aorta

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