This work was supported by Interdisciplinary Centre for Clinical Research IZKF Aachen (junior research group to E.A.L.) within the faculty of Medicine at RWTH Aachen University. We also thank Mrs. Roya Soltan for help with the immunohistochemistry staining.

Experiments presented in this paper are performed according to the German law and to the European animal care guidelines. The animals are bred in the animal facility of the Institute for Laboratory Animal Science, University Hospital Aachen, Germany, under supervision of Prof. Dr. R. Tolba and Dr. A. Teubner (animal welfare officer).
1. Animal Care
<ol>
	<li>Keep the mice in a specialized care unit, ensuring proper access to food and specialized veterinary control and treatment. If the animals are moved or purchased from third parties, please ensure a one week accommodation period before undergoing the procedure.</li>
</ol>
2. Hyperlipidemia Inducement
<ol>
	<li>Feed 6 - 8 week old, 18 - 20 g, female (optionally) ApoE-/- mice with an atherogenic diet (21% fat, 0.15% cholesterol, 19.5% casein, wt/wt) one week prior to the surgical procedure and continue the diet until the atherosclerotic plaque analysis is to be performed.</li>
</ol>
3. Surgical Preparation
<ol>
	<li>Anesthetize the mice using an intraperitoneal injection of 100 mg/kg ketamine by bodyweight and 10 mg/kg xylazine by bodyweight. Confirm proper anesthetization prior to surgery by the lack of reflexes and whisker movement. Place a small amount of sterile eye ointment in the eye to minimize drying.</li>
	<li>Ensure the maintenance of sterile conditions to avoid infections during surgery by using sterile materials and instruments.</li>
	<li>Shave the mice in the ventral neck region. Disinfect the skin with betadine prior to incision. Make a 1 cm skin incision in the median region of the neck area, on top of the trachea.</li>
	<li>Separate the two fat bodies to ensure a proper view over the tracheal region. Use retractors to hold the muscle layer and expose the carotid artery. If present, perform the blunt-dissection of the thin muscle layer covering the carotid artery.</li>
	<li>Use sharp curved forceps to separate the carotid artery from the vagus nerve and jugular vein. Thus, the bifurcation area with the internal and external carotid artery should be visible. Use 0.9% NaCl in order to avoid tissue dryness during the surgical procedure.</li>
</ol>
4. Wire-Injury
<ol>
	<li>Place a 7 cm long 0/5 silk suture under the carotid artery, proximal to the aortic arch. Make an open loop, ready to be closed at any time.</li>
	<li>Place two 0/7 silk sutures (each 1.5 cm long) around the external carotid artery: one loop close to the bifurcation point, and one loop as distal as possible. Prepare them as an open loop, ready to be closed at any time.</li>
	<li>Place one 0/7 silk suture (1.5 cm long) under the internal carotid artery. Prepare it as an open loop, ready to be closed at any time.</li>
	<li>Position the mouse table with the mouse head towards the operator to ensure proper positioning for the guide-wire insertion during the denudation (Figure 1A).</li>
	<li>Under the microscopic view, stop the blood flow through the common carotid artery by holding and pulling the ends of the 0/5 silk suture with hemostat forceps.</li>
	<li>Immediately after the common carotid artery ligature, close the suture loops placed on the internal carotid artery and the distal suture on the external carotid artery tightly (Figure 1B).</li>
	<li>Perform a small incision (arteriotomy, half of the vessel diameter) distal to the external carotid artery, between the two loops, using small scissors (Figure 1C). If the incision is too big, please follow the troubleshooting instructions (see the Discussion).</li>
	<li>Use commercially polished guide wires or use in-house specialized personnel to polish the guide wires. Disinfect the 14 inch polished flexible guide-wire with alcohol and moisten it in a droplet of 0.9% NaCl to ensure proper sliding into the vessel.</li>
	<li>Insert the guide-wire into the common carotid artery via the transverse arteriotomy of the external carotid artery (Figure 1D). Obtain endothelial denudation by passing the guide wire along the vessel while rotating. Repeat this procedure three times. Maintain the same amplitude of rotational movement in each mouse to increase the reproducibility.</li>
	<li>Close the proximal loop on the external carotid artery tightly. Restore the blood flow in the carotid artery by cutting the suture around common artery and the suture around the internal carotid artery.</li>
</ol>
5. Suture and Recovery
<ol>
	<li>Remove the retractors and return the muscle layer and the two fat bodies to the physiological position.</li>
	<li>Close the skin with three separated sutures 0/6, if echocardiographic measurements are needed. If no imaging is needed, use metallic clips to close the skin.</li>
	<li>Lay the mouse down on its left side under the infrared light until it wakes up. Do not leave an animal unattended nor in the company of other animals until fully recovered.</li>
	<li>For future identification, mark the mouse using the local system. Ask the animal welfare officer from the local institution.</li>
</ol>
6. Analysis of the Atherosclerotic Plaque
<ol>
	<li>Anesthetize the mice at the end-time point using an intraperitoneal injection of 100 mg/kg ketamine by bodyweight and 10 mg/kg xylazine by bodyweight. Confirm proper anesthetization by the lack of reflexes and whisker movement.</li>
	<li>Perform exsanguination by retro-orbital or cardiac puncture and collect the blood for further analysis2.</li>
	<li>Disinfect the skin with betadine. Open the thoracic cavity and remove the right auriculum of the heart. Perfuse phosphate buffered solution through left ventricle to remove the remaining blood from the vessel and then perfuse 4% PFA to fix the tissue.</li>
	<li>If no fixation is required, explant the carotid artery immediately after washing2,4,11. Perform standard protocols with analyses of interest: paraffin embedding, cryosection, mRNA or protein analysis, etc.</li>
	<li>For morphometrical measurements, carefully explant the carotid artery including the bifurcation, with minimal manipulation, as proximal to the aortic arch using curved forceps and small scissors.</li>
	<li>Embed the carotid artery in the paraffin block using standard embedding protocols. To perform transversal sectioning, place the carotid artery upright on bifurcation. Cut 5 &#181;m thick serial sections starting with the bifurcation and collect them all on coated histological slides (Figure 2A).</li>
	<li>Stain every 10th section using Movat staining to highlight the laminas2,4,11. After collecting microscopic pictures of all vessels (using a 10X objective), measure the lumen, as well as the internal and external lamina for each section, using special designed software2,4,11, as shown in Figure 2B. Calculate the intimal growth and media of the vessels.</li>
	<li>Analyze smooth muscle cells and macrophage content, or endothelial recovery in serial sections, using usual immunohistological staining2 (Figure 2C).</li>
</ol>

<ol><li>Simsekyilmaz, S., Liehn, E. A., Militaru, C., Vogt, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Progress+in+interventional+cardiology%3A+challenges+for+the+future%5BTitle%5D)+AND+%22Thromb+Haemost%22%5BJournal%5D)">Progress in interventional cardiology: challenges for the future.</a> Thromb Haemost. 113 (3), 464-472 (2015).</li>
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<li>Schwartz, R. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Preclinical+evaluation+of+drug-eluting+stents+for+peripheral+applications%3A+recommendations+from+an+expert+consensus+group%5BTitle%5D)+AND+%22Circulation%22%5BJournal%5D)">Preclinical evaluation of drug-eluting stents for peripheral applications: recommendations from an expert consensus group.</a> Circulation. 110 (16), 2498-2505 (2004).</li>
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<li>Liehn, E. A., Schober, A., Weber, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Blockade+of+keratinocyte-derived+chemokine+inhibits+endothelial+recovery+and+enhances+plaque+formation+after+arterial+injury+in+ApoE-deficient+mice%5BTitle%5D)+AND+%22Arterioscler+Thromb+Vasc+Biol%22%5BJournal%5D)">Blockade of keratinocyte-derived chemokine inhibits endothelial recovery and enhances plaque formation after arterial injury in ApoE-deficient mice.</a> Arterioscler Thromb Vasc Biol. 24 (10), 1891-1896 (2004).</li>
<li>Liehn, E. A., Zernecke, A., Postea, O., Weber, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Chemokines%3A+inflammatory+mediators+of+atherosclerosis%5BTitle%5D)+AND+%22Arch+Physiol+Biochem%22%5BJournal%5D)">Chemokines: inflammatory mediators of atherosclerosis.</a> Arch Physiol Biochem. 112 (4-5), 229-238 (2006).</li>
<li>Simsekyilmaz, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Role+of+extracellular+RNA+in+atherosclerotic+plaque+formation+in+mice%5BTitle%5D)+AND+%22Circulation%22%5BJournal%5D)">Role of extracellular RNA in atherosclerotic plaque formation in mice.</a> Circulation. 129 (5), 598-606 (2014).</li>
<li>Wu, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Rhodamine-loaded+intercellular+adhesion+molecule-1-targeted+microbubbles+for+dual-modality+imaging+under+controlled+shear+stresses%5BTitle%5D)+AND+%22Circ+Cardiovasc+Imaging%22%5BJournal%5D)">Rhodamine-loaded intercellular adhesion molecule-1-targeted microbubbles for dual-modality imaging under controlled shear stresses.</a> Circ Cardiovasc Imaging. 6 (6), 974-981 (2013).</li>
<li>Schober, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Crucial+role+of+the+CCL2%2FCCR2+axis+in+neointimal+hyperplasia+after+arterial+injury+in+hyperlipidemic+mice+involves+early+monocyte+recruitment+and+CCL2+presentation+on+platelets%5BTitle%5D)+AND+%22Circ+Res%22%5BJournal%5D)">Crucial role of the CCL2/CCR2 axis in neointimal hyperplasia after arterial injury in hyperlipidemic mice involves early monocyte recruitment and CCL2 presentation on platelets.</a> Circ Res. 95 (11), 1125-1133 (2004).</li>
<li>Ali, Z. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Increased+in-stent+stenosis+in+ApoE+knockout+mice%3A+insights+from+a+novel+mouse+model+of+balloon+angioplasty+and+stenting%5BTitle%5D)+AND+%22Arterioscler+Thromb+Vasc+Biol%22%5BJournal%5D)">Increased in-stent stenosis in ApoE knockout mice: insights from a novel mouse model of balloon angioplasty and stenting.</a> Arterioscler Thromb Vasc Biol. 27 (4), 833-840 (2007).</li>
<li>Chamberlain, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+novel+mouse+model+of+in+situ+stenting%5BTitle%5D)+AND+%22Cardiovasc+Res%22%5BJournal%5D)">A novel mouse model of in situ stenting.</a> Cardiovasc Res. 85, 38-44 (2010).</li>
<li>Rodriguez-Menocal, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+novel+mouse+model+of+in-stent+restenosis%5BTitle%5D)+AND+%22Atherosclerosis%22%5BJournal%5D)">A novel mouse model of in-stent restenosis.</a> Atherosclerosis. 209 (2), 359-366 (2010).</li>
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There are no disclosures by the authors.

In this paper, we provide useful tips to perform the wire-injury procedure even by personnel with minimal experience in animal surgeries. There are two critical steps in performing this procedure: the incision of the external carotid artery and the insertion of the wire. The incision in the external carotid artery needs to be performed as far as possible from the bifurcation, in order to ensure enough remaining material (Figure 1C). The incision should not be too large, due to the risk of cutting the entire vessel. The second critical step is the high risk of bleeding during the arteriotomy and the insertion of the guide wire if blood flow is not efficiently discontinued. Moreover, endothelial denudation might not take place or arterial rupture is possible if the guide wire is not properly introduced in the lumen vessel. To avoid this, the surface of the guide wire must be carefully polished before the operation.
To optimize the protocol, the position of the operating-table with the mouse-head towards the surgeon ensures a better view, accessibility and control for the proper guide wire manipulation. Moreover, to increase the reproducibility, use the same guide wire in all of the studies. Since the wire size does not change, it is important to consider and eliminate all the possible differences between the mice by using the same gender, age and weight for all mice included in a study. Thereafter, Evans-Blue staining will help the surgeon determine the efficiency of the denudation. The existence of appropriate equipment is a prerequisite for the success of the procedure. A 10X stereomicroscope is essential for performing this procedure. The proper preparation of the guide-wire (for example polishing it) is crucial. Therefore, we strongly recommend that the guide wire preparation be performed by specialized technical personnel where available.
There are many troubleshooting steps in this protocol. If incising the external carotid artery near the bifurcation, carefully bind the externa, near the bifurcation, so no bleeding occurs. During cutting, the external carotid artery cannot be seen. Therefore, consider the bifurcation at the level of silk suture. Collect sections when the silk suture disappears. If the incision in the external carotid artery is too large and the vessel is ruptured, ensure that the blood flow into the carotid communis and internal carotid artery is effectively interrupted and try to find the opening of the vessel using forceps. After introducing the guide wire and performing the denudation, bind the vessel near the bifurcation. During cutting, start to collect when the silk from the suture starts to disappear. If arterial rupture occurs during the denudation with the guide wire, check under the microscope if the guide wire is properly polished.
Despite the similarity of the wire-injury model to clinical situations, many groups are focused on native atherosclerosis in mice, or they choose invasive atherosclerosis inductions, such as balloon angioplasty in rats or rabbits, because of the lack of trained personnel who can perform small animal surgeries. Despite the benefits of using rabbits/rats, e.g. no need for miniaturized equipment, neither rat models nor rabbit models offer a variety of different knock-out strains, in terms of studying molecular mechanisms involved in neointima growth and in-stent thrombosis.
The existing models for studying in-stent restenosis in mice are difficult, require high surgical skills, and have high risks of complications such as bleeding or paralysis. For example, the mechanical injury or stent-implantation into the thoracic aorta via femoral artery is accompanied by a high mortality rate (35%) due to hind leg paralysis or bleeding13-15. We also describe stent implantation in the carotid artery of a mouse16. The procedure is similar; however, the tissue processing for analysis is complicated and is not available to all laboratories16. The carotid artery is directly accessible, not only for operation procedures, but also for existing imaging methods such as ultrasound imaging. Other injury inductions in the carotid arteries in mice can be done using electrical devices17. This method is easy to perform and ensures high reproducibility. However, it induces injury in all vessel layers, which is not identical with mechanical injury. Balloon applications have benefits, e.g. the adjustment to the vessel diameter in line with the clinical practice and has strong influence on the pathological outcome. Even though mouse balloons are available, they are very expensive and therefore, not widely used. Instead, the wire-injury is the established method, mimicking in-stent stenosis.
The denudation is performed on the normal arterial wall, though with an atherosclerotic background. Therefore, the neointima formation will be moderate compared to the clinical situation. The high number of preclinical models demonstrates that none of the models fulfill all of the criteria necessary to uncover the entirety of the cellular and molecular mechanisms leading to the pathophysiology in humans (see Table 2).
After performing the wire-injury procedure, other biological and molecular analysis can be performed to identify cells, proteins, mRNAs, microRNAs, genes or other biomarkers, which can be used as therapeutic targets to develop new treatment strategies for atherosclerosis, and in particular for neointima formation after vascular injury. If available, the plaque growth can be monitored using high frequency ultrasound or other high-resolution imaging techniques. Moreover, mastering this technique would give the operator the opportunity to adapt the protocol to other invasive atherosclerosis inducement models, such as collar placement, partial ligation or even stent implantation.

Atherosclerosis is the main pathology underlying cardiovascular events such as myocardial infarction or stroke. The main mechanisms triggering acute cardiovascular syndromes are plaque rupture, superficial erosion, and thrombus formation. There are multiple clinical situations connected to the plaque development: native atherosclerotic plaque, restenosis after endarterectomy, and restenosis after balloon angioplasty with/without stent implantation1. After arterial injury, suppression of the inflammatory processes2,3 and the recovery of the endothelial compartment are essential to prevent further complications1. Clinical research is limited to tissue and blood samples due to ethical considerations, costs, and a lack of knowledge in basic mechanisms. For these reasons, there is a need to study molecular mechanisms in animal models4-6, which can recreate the clinical conditions. Our model of accelerated neointima formation in the context of atherosclerosis is the result of many years of experience in the implementation of these models in small animals7-11. The mouse model is the most attractive model for research, due to its ease of handling, the ability to have large animal groups due to low costs related to animal purchase and care, and the availability of various transgenic and knockout strains.
The major disadvantage of the mouse model is the small size of the main arteries subjected to atherosclerotic disease (the carotid artery, the aorta, and the femoral artery), which requires qualified surgical expertise and skills to manipulate the vessels and to invasively induce an atherosclerotic plaque. Therefore, the model of accelerated neointima formation, in the context of restenosis after endarterectomy or stent implantation, proposed in this paper is presented with a step-by-step guideline and suggestions to ease the introduction for interested personnel. Another disadvantage is that the denudation is made on the normal arterial wall, and therefore, the neo-intima formation will be moderate compared with the clinical situation. The high level of plasma cholesterol reached in apolipoprotein E knockout (Apoe-/-) mice fed with a high fat-diet creates a proper pro-inflammatory environment needed for the neo-intima formation.
The surgery is performed under a stereomicroscope. The carotid artery is exposed by a median incision in the ventral cervical area. Anatomical structures on top of and surrounding the carotid artery are minimally manipulated to reduce post-surgical inflammation. The carotid artery bifurcation is exposed. To induce accelerated neointima formation, internal and external carotid arteries are prepared for blood flow cessation and subsequent common carotid artery denudation. In conclusion, the method can be learned by personnel with minimal experience in animal surgeries.

<table><tbody><tr><td>Stereomicroscope</td><td>Olympus</td><td>SZ/X9</td><td>-</td></tr><tr><td>Forceps</td><td>FST, Germany</td><td>91197-00</td><td>standard tip curved 0,17 mm</td></tr><tr><td>Hemostat forceps</td><td>FST, Germany</td><td>13007-12</td><td>curved</td></tr><tr><td>Scissors</td><td>FST, Germany</td><td>91460-11</td><td>Straight</td></tr><tr><td>Vannas scissor</td><td>Aesculap, Germany</td><td>OC 498 R</td><td>-</td></tr><tr><td>Retractors</td><td>FST, Germany</td><td>18200-10</td><td>2.5 mm wide</td></tr><tr><td>Retractors</td><td>FST, Germany</td><td>18200-11</td><td>5 mm wide</td></tr><tr><td>Ketamine 10%</td><td>CEVA, Germany</td><td>-</td><td>-</td></tr><tr><td>Xylazine 2%</td><td>Medistar, Germany</td><td>-</td><td>-</td></tr><tr><td>Bepanthene eye and nose cream</td><td>Bayer, Germany</td><td>-</td><td>-</td></tr><tr><td>Silicon tube</td><td>IFK Isofluor, Germany</td><td>custom-made&amp;nbsp; product</td><td>diameter 500 &amp;micro;m,</td></tr><tr><td>section thickness 100 &amp;micro;m,</td><td></td><td></td><td></td></tr><tr><td>polytetrafluorethylene catheter</td><td></td><td></td><td></td></tr><tr><td>PROLENE Suture 6/0&amp;nbsp;</td><td>ETHICON</td><td>8707H&amp;nbsp;</td><td>polypropylene monofilament suture, unresorbable, needle CC-1, 13 mm, 3/8 Circle&amp;nbsp;</td></tr><tr><td>7/0 Silk</td><td>Seraflex</td><td>IC 1005171Z</td><td>-</td></tr><tr><td>Michel Suture Clips</td><td>FST, Germany</td><td>12040-01&amp;nbsp;</td><td>&amp;nbsp;-&amp;nbsp;</td></tr><tr><td>Clip Applying Forcep</td><td>FST, Germany</td><td>12018-12&amp;nbsp;</td><td>&amp;nbsp;-&amp;nbsp;</td></tr><tr><td>14&amp;rdquo;Wire for Catheter</td><td>Abbot</td><td>1000462H</td><td>Use 10 cm from stiff part and equalize the ends</td></tr><tr><td>Mice</td><td>Charles River</td><td>Apolipoprotein E -/- mice with C57/Bl6 background</td><td>&amp;nbsp;-&amp;nbsp;</td></tr></tbody></table>

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.

The atherosclerotic plaque induction procedure takes 15 - 20 min and shows a minimal mortality rate, mostly due to the bleeding occurring during the procedure. After surgery, the mice recover from anesthesia within 20 - 25 min. No physical impairment, such as paralysis, or feeding disturbance was observed after the surgery.
The wire-injury induces a de-endothelialization, mimicking vascular lesions after balloon denudation or stent-implantation. Immediately after injury, the denuded vascular wall will be covered with a layer of thrombocytes, which mediates and favors the adhesion of the monocytes12. Activated smooth muscle cells from the media will proliferate and migrate into the intimal spaces, forming the neointima. Other progenitors for smooth muscle cells will migrate from the blood (estimated to be 40%) and contribute to the neointima growth. The plaque formation will end after the complete re-endothelialization, usually 4 weeks after the wire-injury.
The neointima formation can be assessed using Movat staining. The plaque size is calculated for each slide using software as shown in Figure 2B. The total plaque size (left carotid artery) can vary between 70,000 - 100,000 &#181;m&#178;, while the control vessel size (right carotid artery) can vary between 7,000 - 8,000 &#181;m&#178;. These values depend largely on the surgeon. Therefore, we strongly recommend using the same surgeon during the experiments for the same study.
The developed plaque resembles in stent restenosis, which predominately consists of proliferated and migrated smooth muscle cells from the media. The cellular composition determined by immunological staining procedures shows that the smooth muscle cell content is approximately 30 - 40%, while macrophages are found in 15 - 25% of the neointima of the injured vessel. The re-endothelialization can be measured after staining for an endothelial marker, and calculated as the percentage of circumference stained over the entire circumference of the lumen. Usually the re-endothelialization reaches 80 - 90% after 3 weeks, and should almost be complete after 4 weeks (Figure 2C). To track the plaque growth during its development, the same analysis can be repeated for every time-point after the wire-injury, depending on the interest and the subject studied (see Table 1).
<img alt="Figure 1" src="/files/ftp_upload/54571/54571fig1.jpg" /> 
Figure 1. Schematic Representation of Operative Procedure. (A) The positioning of the operation table toward the operator during the wire-injury procedure (B) Enlarged view of the common carotid artery and its branches, as it appears under the microscope at 10X magnification (C) The size of the incision in the external carotid artery under the microscope at 10X magnification (D) Schematic representation of wire-injury procedure using the 14 inch guide wire. <a href="http://cloudflare.jove.com/files/ftp_upload/54571/54571fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54571/54571fig2.jpg" /> 
Figure 2. Analysis of Restenosis Plaque. (A) Schematic representation of plaque analysis in the common carotid artery, 4 weeks after wire-injury induction (B) Neointima formation 4 weeks after the wire-injury and schematic representation of main parameters used for analysis. Intima (green area) is the difference between the lumen (red) and the lamina interna (green line). Media (yellow area) is the difference between the lamina externa (yellow line) and interna (green line). Scale bar 100 &#181;m (C) Representative images of the staining of the main cell types involved in neointima formation. Smooth muscle cells (smooth muscle actin -red, scale bar 100 &#181;m), macrophages (Mac 2- green, scale bar 100 &#181;m) and endothelial cells (CD31- red, arrows, scale bar 50 &#181;m). <a href="http://cloudflare.jove.com/files/ftp_upload/54571/54571fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Time</td>
			<td>Trombus</td>
			<td>Plaque (&#181;m&#178;)</td>
			<td>Macrophages 
			(% from Plaque)</td>
			<td>Smooth muscle cells 
			(% from Plaque)</td>
			<td>Re-endothelialization 
			(% lumen circumference)</td>
		</tr>
		<tr>
			<td>1 day</td>
			<td>present</td>
			<td>0</td>
			<td>0</td>
			<td>0</td>
			<td>0</td>
		</tr>
		<tr>
			<td>1 week</td>
			<td>-</td>
			<td>&#60; 30 000</td>
			<td>&#62; 10</td>
			<td>&#60; 50</td>
			<td>&#60; 50</td>
		</tr>
		<tr>
			<td>2 weeks</td>
			<td>-</td>
			<td>&#60; 50 000</td>
			<td>&#62; 10</td>
			<td>&#60; 50</td>
			<td>&#62; 50</td>
		</tr>
		<tr>
			<td>3 weeks</td>
			<td>-</td>
			<td>&#60; 70 000</td>
			<td>15-25</td>
			<td>30-40</td>
			<td>80-90</td>
		</tr>
		<tr>
			<td>4 weeks</td>
			<td>-</td>
			<td>70 000 &#8211; 100 000</td>
			<td>15-25</td>
			<td>30-40</td>
			<td>complete</td>
		</tr>
	</tbody>
</table>
Table 1. Time-dependent Plaque&#39;s Development.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Model</td>
			<td>Animals</td>
			<td>Advantages</td>
			<td>Disanvantages</td>
		</tr>
		<tr>
			<td rowspan="2">Diet-induced native atherosclerosis</td>
			<td>Small</td>
			<td>
			<ul>
				<li>mimics the atherosclerosis pathology</li>
				<li>ease of handling</li>
				<li>no surgery</li>
				<li>no stress for the animals</li>
				<li>low costs related to animal purchase and care</li>
				<li>availability of various transgenic and knockout strains</li>
			</ul>
			</td>
			<td>
			<ul>
				<li>low reproductibility</li>
				<li>high variance</li>
				<li>increased animal&#8217;s&#160; number required</li>
				<li>increased waiting time</li>
			</ul>
			</td>
		</tr>
		<tr>
			<td>Big</td>
			<td>
			<ul>
				<li>mimics the atherosclerosis pathology</li>
				<li>ease of handling</li>
				<li>no surgery</li>
				<li>no stress for the animals</li>
			</ul>
			</td>
			<td>
			<ul>
				<li>low reproductibility</li>
				<li>high variance</li>
				<li>increased animal&#8217;s number required</li>
			</ul>
			</td>
		</tr>
		<tr>
			<td rowspan="2">Balloon dilatation</td>
			<td>Small</td>
			<td>
			<ul>
				<li>mimics restenosis after balloon angioplasty</li>
				<li>low costs related to animal purchase and care</li>
				<li>availability of various transgenic and knockout strains</li>
			</ul>
			</td>
			<td>
			<ul>
				<li>small size of the main arteries</li>
				<li>requires qualified surgical expertise</li>
				<li>balloons very expensive</li>
				<li>denudation is made on the normal arterial wall</li>
				<li>existence of appropriate equipment</li>
				<li>risks of complications as bleeding or paralysis</li>
			</ul>
			</td>
		</tr>
		<tr>
			<td>Big</td>
			<td>
			<ul>
				<li>mimics restenosis after balloon angioplasty</li>
				<li>&#160; ease of handling</li>
				<li>use of devices for humans</li>
			</ul>
			</td>
			<td>
			<ul>
				<li>denudation is made on the normal arterial wall</li>
			</ul>
			</td>
		</tr>
		<tr>
			<td>Wire Injury</td>
			<td>Small</td>
			<td>
			<ul>
				<li>mimics restenosis after balloon angioplasty</li>
				<li>ease of handling</li>
				<li>minimal mortality rate</li>
				<li>low costs related to animal purchase and care</li>
				<li>availability of various transgenic and knockout strains</li>
				<li>no physical impairment</li>
			</ul>
			</td>
			<td>
			<ul>
				<li>small size of the main arteries</li>
				<li>requires less qualified surgical expertise</li>
				<li>denudation is made on the normal arterial wall</li>
				<li>existence of appropriate equipment</li>
			</ul>
			</td>
		</tr>
		<tr>
			<td rowspan="2">Stent implantation</td>
			<td>Small</td>
			<td>
			<ul>
				<li>&#160; mimics restenosis and thrombosis after stent implantation</li>
				<li>low costs related to animal purchase and care</li>
				<li>availability of various transgenic and knockout strains</li>
			</ul>
			</td>
			<td>
			<ul>
				<li>small size of the main arteries</li>
				<li>requires qualified surgical expertise</li>
				<li>small stents not available</li>
				<li>denudation is made on the normal arterial wall</li>
				<li>increased mortality</li>
				<li>existence of appropriate equipment</li>
				<li>risks of complications as bleeding or paralysis</li>
			</ul>
			</td>
		</tr>
		<tr>
			<td>Big</td>
			<td>
			<ul>
				<li>mimics restenosis 
				and thrombosis after stent implantation</li>
				<li>&#160; ease of handling</li>
				<li>use of devices for humans</li>
			</ul>
			</td>
			<td>
			<ul>
				<li>denudation is made on the normal arterial wall</li>
			</ul>
			</td>
		</tr>
	</tbody>
</table>
Table 2. Advantages and Disadvantages of Existent Models of Arterial Injury.

Watch this Scientific Journal Video about Induction of Accelerated Atherosclerosis in Mice: The "Wire-Injury" Model at JoVE.com

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

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10.8K Views.  RWTH Aachen University. 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. 

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

Implantation of a Carotid Cuff for Triggering Shear-stress Induced Atherosclerosis in Mice

It is widely accepted that alterations in vascular shear stress trigger the expression of inflammatory genes in endothelial cells and thereby induce atherosclerosis (reviewed in 1 and 2). The role of shear stress has been extensively studied in vitro investigating the influence of flow dynamics on cultured endothelial cells 1,3,4 and in vivo in larger animals and humans 1,5,6,7,8. However, highly reproducible small animal models allowing systematic investigation of the influence of shear stress on plaque development are rare. Recently, Nam et al. 9 introduced a mouse model in which the ligation of branches of the carotid artery creates a region of low and oscillatory flow. Although this model causes endothelial dysfunction and rapid formation of atherosclerotic lesions in hyperlipidemic mice, it cannot be excluded that the observed inflammatory response is, at least in part, a consequence of endothelial and/or vessel damage due to ligation.
In order to avoid such limitations, a shear stress modifying cuff has been developed based upon calculated fluid dynamics, whose cone shaped inner lumen was selected to create defined regions of low, high and oscillatory shear stress within the common carotid artery 10. By applying this model in Apolipoprotein E (ApoE) knockout mice fed a high cholesterol western type diet, vascular lesions develop upstream and downstream from the cuff. Their phenotype is correlated with the regional flow dynamics 11 as confirmed by in vivo Magnetic Resonance Imaging (MRI) 12: Low and laminar shear stress upstream of the cuff causes the formation of extensive plaques of a more vulnerable phenotype, whereas oscillatory shear stress downstream of the cuff induces stable atherosclerotic lesions 11. In those regions of high shear stress and high laminar flow within the cuff, typically no atherosclerotic plaques are observed.
 
In conclusion, the shear stress-modifying cuff procedure is a reliable surgical approach to produce phenotypically different atherosclerotic lesions in ApoE-deficient mice.

The constricting cuff presented in this article is designed to induce atherosclerosis in the murine common carotid artery. Due to the conical shape of its inner lumen the implanted cuff generates well-defined regions of low, high and oscillatory shear stress triggering the development of atherosclerotic lesions of different inflammatory phenotypes.

It is widely accepted that alterations in vascular shear stress trigger the expression of inflammatory genes in endothelial cells and thereby induce ...

Generation and 3-Dimensional Quantitation of Arterial Lesions in Mice Using Optical Projection Tomography

The generation and analysis of vascular lesions in appropriate animal models is a cornerstone of research into cardiovascular disease, generating important information on the pathogenesis of lesion formation and the action of novel therapies. Use of atherosclerosis-prone mice, surgical methods of lesion induction, and dietary modification has dramatically improved understanding of the mechanisms that contribute to disease development and the potential of new treatments.
Classically, analysis of lesions is performed ex vivo using 2-dimensional histological techniques. This article describes application of optical projection tomography (OPT) to 3-dimensional quantitation of arterial lesions. As this technique is non-destructive, it can be used as an adjunct to standard histological and immunohistochemical analyses.
Neointimal lesions were induced by wire-insertion or ligation of the mouse femoral artery whilst atherosclerotic lesions were generated by administration of an atherogenic diet to apoE-deficient mice.
Lesions were examined using OPT imaging of autofluorescent emission followed by complementary histological and immunohistochemical analysis. OPT clearly distinguished lesions from the underlying vascular wall. Lesion size was calculated in 2-dimensional sections using planimetry, enabling calculation of lesion volume and maximal cross-sectional area. Data generated using OPT were consistent with measurements obtained using histology, confirming the accuracy of the technique and its potential as a complement (rather than alternative) to traditional methods of analysis.
This work demonstrates the potential of OPT for imaging atherosclerotic and neointimal lesions. It provides a rapid, much needed ex vivo technique for the routine 3-dimensional quantification of vascular remodelling.

Ex vivo analysis of arterial lesions from animal models of cardiovascular disease classically relies on histological and immunohistochemical techniques. These provide 2-dimensional measurements in 3-dimensional lesions. This manuscript describes the generation of arterial lesions for quantitative analysis in 3-dimensions using optical projection tomography.

The generation and analysis of vascular lesions in appropriate animal models is a cornerstone of research into cardiovascular disease, generating ...

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 ...

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. ...

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 ...

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 ...

Induction of Atherosclerotic Plaques Through Activation of Mineralocorticoid Receptors in Apolipoprotein E-deficient Mice

Atherosclerosis is due to a chronic inflammatory response affecting vascular endothelium and is promoted by several factors such as hypertension, dyslipidemia, and diabetes. To date, there is evidence to support a role for circulating aldosterone as a risk factor for the development of cardiovascular disease. Transgenic mouse models have been generated to study cellular and molecular processes leading to atherosclerosis. In this manuscript, we describe a protocol that takes advantage of continuous infusion of aldosterone in ApoE-/- mice and generates atherosclerotic plaques in the aortic root after 4 weeks of treatment. We, therefore, illustrate a method for quantification and characterization of atherosclerotic lesions at the aortic root level. The added value of aldosterone infusion is represented by the generation of atherosclerotic lesions rich in lipid and inflammatory cells after 4 weeks of treatment. We describe in detail the staining procedures to quantify lipid and macrophage content within the plaque. Notably, in this protocol, we perform heart tissue-embedding in OCT in order to preserve the antigenicity of cardiac tissue and facilitate detectability of antigens of interest. Analysis of the plaque phenotype represents a valid approach to study the pathophysiology of atherosclerosis development and to identify novel pharmacological targets for the development of anti-atherogenic drugs.

We describe a protocol to induce atherosclerosis in the aortic root of ApoE-/- mice fed with an atherogenic diet, through a continuous release of aldosterone. Methods to characterize plaque composition are also described.

Atherosclerosis is due to a chronic inflammatory response affecting vascular endothelium and is promoted by several factors such as hypertension, ...

A Modified Simple Method for Induction of Myocardial Infarction in Mice

Myocardial infarction (MI) represents one of the leading causes of death. MI models are widely used for investigating the pathomechanisms of post-MI remodeling and evaluation of novel therapeutics. Different methods (e.g., isoproterenol treatment, cryoinjury, coronary artery ligation, etc.) have been used to induce MI. Compared with isoproterenol treatment and cryoinjury, coronary artery ligation may better reflect the ischemic response and chronic remodeling after MI. However, traditional methods for coronary ligation in mice are technically challenging. The current study describes a simple and efficient process for induction of MI in mice with readily available materials. The mouse chest skin was cut open under stable anesthesia. The heart was immediately externalized through the intercostal space after blunt separation of the pectoralis major and pectoralis minor. The left anterior descending branch (LAD) was ligated with a 6-0 suture 3 mm from its origin. Following LAD ligation, staining with 2,3,5-Triphenyltetrazolium chloride (TTC) indicated successful induction of MI and temporal changes of post-MI scar size. Meanwhile, survival analysis results showed overt mortality within 7 days after MI, mainly due to cardiac rupture. Moreover, post-MI echocardiographic assessment demonstrated successful induction of contractile dysfunction and ventricular remodeling. Once mastered, an MI model can be established in mice within 2-3 min with readily available materials.

Under adequate anesthesia, the mouse heart was externalized through the intercostal space, and myocardial infarction was successfully induced by ligating the left anterior descending artery (LAD) using materials readily available in most laboratories.

Myocardial infarction (MI) represents one of the leading causes of death. MI models are widely used for investigating the pathomechanisms of post-MI ...

Induction of Myocardial Infarction and Myocardial Ischemia-Reperfusion Injury in Mice

Acute myocardial infarction is a common cardiovascular disease with high mortality. Myocardial reperfusion injury can counteract the beneficial effects of heart reflow and induce secondary myocardial injury. A simple and reproducible model of myocardial infarction and myocardial ischemia-reperfusion injury is a good tool for researchers. Here, a customizable method to create a myocardial infarction (MI) model and MIRI by precision ligation of the left anterior descending coronary artery (LAD) through micromanipulation is described. Accurate and reproducible ligature positioning of the LAD helps obtain consistent results for heart injury. ST-segment changes can help to identify model accuracy. The serum level of cardiac troponin T (cTnT) is used to assess the myocardial injury, cardiac ultrasound is employed to evaluate the myocardial systolic function, and Evans-Blue/triphenyl tetrazolium chloride staining is used to measure infarct size. In general, this protocol reduces procedure duration, ensures controllable infarct size, and improves mouse survival.

Here we describe a simple and reproducible method that can induce myocardial infarction or myocardial ischemia-reperfusion injury in mice by precision ligation of the left anterior descending coronary artery through micromanipulation.

Acute myocardial infarction is a common cardiovascular disease with high mortality. Myocardial reperfusion injury can counteract the beneficial effects of ...

Occlusion of Distal Middle Artery to Develop an Ischemic Stroke Mouse Model

Induction of Acute Ischemic Stroke in Mice Using the Distal Middle Artery Occlusion Technique

Ischemic stroke remains the predominant cause of mortality and functional impairment among the adult populations globally. Only a minority of ischemic stroke patients are eligible to receive intravascular thrombolysis or mechanical thrombectomy therapy within the optimal time window. Among those stroke survivors, around two-thirds suffer neurological dysfunctions over an extended period. Establishing a stable and repeatable experimental ischemic stroke model is extremely significant for further investigating the pathophysiological mechanisms and developing effective therapeutic strategies for ischemic stroke. The middle cerebral artery (MCA) represents the predominant location of ischemic stroke in humans, with the MCA occlusion serving as the frequently employed model of focal cerebral ischemia. In this protocol, we describe the methodology of establishing the distal MCA occlusion (dMCAO) model through transcranial electrocoagulation in C57BL/6 mice. Since the occlusion site is located at the cortical branch of MCA, this model generates a moderate infarcted lesion restricted to the cortex. Neurological behavioral and histopathological characterization have demonstrated visible motor dysfunction, neuron degeneration, and pronounced activation of microglia and astrocytes in this model. Thus, this dMCAO mouse model provides a valuable tool for investigating the ischemiastroke and worth of popularization.

Author Spotlight: Establishing a Reliable Distal MCA Occlusion Model in Mice for Stroke Research

Here, we present a protocol to establish a distal middle cerebral artery occlusion (dMCAO) model through transcranial electrocoagulation in C57BL/6J mice and evaluate the subsequent neurological behavior and histopathological features.

Ischemic stroke remains the predominant cause of mortality and functional impairment among the adult populations globally. Only a minority of ischemic ...