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>

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A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+in-stent+stenosis+in+ApoE+knockout+mice:+insights+from+a+novel+mouse+model+of+balloon+angioplasty+and+stenting.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+mouse+model+of+in+situ+stenting.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+mouse+model+of+in-stent+restenosis.">A novel mouse model of in-stent restenosis.</a> Atherosclerosis. 209 (2), 359-366 (2010).</li><li>Simsekyilmaz, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+murine+model+of+stent+implantation+in+the+carotid+artery+for+the+study+of+restenosis.">A murine model of stent implantation in the carotid artery for the study of restenosis.</a> J Vis Exp. , e50233 (2013).</li><li>Schroder, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NADPH+oxidase+Nox2+is+required+for+hypoxia-induced+mobilization+of+endothelial+progenitor+cells.">NADPH oxidase Nox2 is required for hypoxia-induced mobilization of endothelial progenitor cells.</a> Circ Res. 105 (6), 537-544 (2009).</li></ol>

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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>2.5mm wide</td>
		</tr>
		<tr>
			<td>Retractors</td>
			<td>FST, Germany</td>
			<td>18200-11</td>
			<td>5mm 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 rowspan="3">Silicon tube</td>
			<td rowspan="3">IFK Isofluor, Germany</td>
			<td rowspan="3">custom-made&#160; product</td>
			<td>diameter 500&#181;m,</td>
		</tr>
		<tr>
			<td>section thickness 100 &#181;m,</td>
		</tr>
		<tr>
			<td>polytetrafluorethylene catheter</td>
		</tr>
		<tr>
			<td>PROLENE Suture 6/0&#160;</td>
			<td>ETHICON</td>
			<td>8707H&#160;</td>
			<td>polypropylene monofilament suture, unresorbable, needle CC-1, 13mm, 3/8 Circle&#160;</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&#160;</td>
			<td>&#160;-&#160;</td>
		</tr>
		<tr>
			<td>Clip Applying Forcep</td>
			<td>FST, Germany</td>
			<td>12018-12&#160;</td>
			<td>&#160;-&#160;</td>
		</tr>
		<tr>
			<td>14&#8221;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>&#160;-&#160;</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="https://cloudflare-jove-com.remotexs.ntu.edu.sg/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="https://cloudflare-jove-com.remotexs.ntu.edu.sg/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. ...

induction-of-accelerated-atherosclerosis-in-mice-the-wire-injury-model

Nanyang Technological University

Research

JoVE Journal

Medicine

10.7K 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 ...

Pharmacologic Induction of Epidermal Melanin and Protection Against Sunburn in a Humanized Mouse Model

Fairness of skin, UV sensitivity and skin cancer risk all correlate with the physiologic function of the melanocortin 1 receptor, a Gs-coupled signaling protein found on the surface of melanocytes. Mc1r stimulates adenylyl cyclase and cAMP production which, in turn, up-regulates melanocytic production of melanin in the skin. In order to study the mechanisms by which Mc1r signaling protects the skin against UV injury, this study relies on a mouse model with "humanized skin" based on epidermal expression of stem cell factor (Scf). K14-Scf transgenic mice retain melanocytes in the epidermis and therefore have the ability to deposit melanin in the epidermis. In this animal model, wild type Mc1r status results in robust deposition of black eumelanin pigment and a UV-protected phenotype. In contrast, K14-Scf animals with defective Mc1r signaling ability exhibit a red/blonde pigmentation, very little eumelanin in the skin and a UV-sensitive phenotype. Reasoning that eumelanin deposition might be enhanced by topical agents that mimic Mc1r signaling, we found that direct application of forskolin extract to the skin of Mc1r-defective fair-skinned mice resulted in robust eumelanin induction and UV protection 1. Here we describe the method for preparing and applying a forskolin-containing natural root extract to K14-Scf fair-skinned mice and report a method for measuring UV sensitivity by determining minimal erythematous dose (MED). Using this animal model, it is possible to study how epidermal cAMP induction and melanization of the skin affect physiologic responses to UV exposure.

Epidermal melanin is induced by topical application of forskolin in a murine model of the fair-skinned UV-sensitive human. Pharmacologic manipulation of cAMP levels in the skin and epidermal darkening strongly protect against UV-mediated inflammation (sunburn) as measured by the minimum erythematous dose (MED) assay.

Fairness of skin, UV sensitivity and skin cancer  risk all correlate with the physiologic function of the melanocortin 1  receptor, a Gs-coupled signaling ...

Induction of Invasive Transitional Cell Bladder Carcinoma in Immune Intact Human MUC1 Transgenic Mice:  A Model for Immunotherapy Development

A preclinical model of invasive bladder cancer was developed in human mucin 1 (MUC1) transgenic (MUC1.Tg) mice for the purpose of evaluating immunotherapy and/or cytotoxic chemotherapy. To induce bladder cancer, C57BL/6 mice (MUC1.Tg and wild type) were treated orally with the carcinogen N-butyl-N-(4-hydroxybutyl)nitrosamine (OH-BBN) at 3.0 mg/day, 5 days/week for 12 weeks. To assess the effects of OH-BBN on serum cytokine profile during tumor development, whole blood was collected via submandibular bleeds prior to treatment and every four weeks. In addition, a MUC1-targeted peptide vaccine and placebo were administered to groups of mice weekly for eight weeks. Multiplex fluorometric microbead immunoanalyses of serum cytokines during tumor development and following vaccination were performed. At termination, interferon gamma (IFN-&#947;)/interleukin-4 (IL-4) ELISpot analysis for MUC1 specific T-cell immune response and histopathological evaluations of tumor type and grade were performed. The results showed that: (1) the incidence of bladder cancer in both MUC1.Tg and wild type mice was 67%; (2) transitional cell carcinomas (TCC) developed at a 2:1 ratio compared to squamous cell carcinomas (SCC); (3) inflammatory cytokines increased with time during tumor development; and (4) administration of the peptide vaccine induces a Th1-polarized serum cytokine profile and a MUC1 specific T-cell response. All tumors in MUC1.Tg mice were positive for MUC1 expression, and half of all tumors in MUC1.Tg and wild type mice were invasive. In conclusion, using a team approach through the coordination of the efforts of pharmacologists, immunologists, pathologists and molecular biologists, we have developed an immune intact transgenic mouse model of bladder cancer that expresses hMUC1.

An N-butyl-N-(4-hydroxybutyl)nitrosamine-induced bladder cancer model was developed in human mucin 1 (MUC1) transgenic mice for the purpose of testing MUC1-directed immunotherapy. After administering a MUC1-targeted peptide vaccine, a cytotoxic T lymphocyte response to MUC1 was confirmed by measuring serum cytokine levels and T-cell specific activity.

A preclinical model of invasive bladder cancer was developed in human mucin 1 (MUC1) transgenic (MUC1.Tg) mice for the purpose of evaluating immunotherapy ...

Quantitative Analysis and Characterization of Atherosclerotic Lesions in the Murine Aortic Sinus

Atherosclerosis is a disease of the large arteries and a major underlying cause of myocardial infarction and stroke. Several different mouse models have been developed to facilitate the study of the molecular and cellular pathophysiology of this disease. In this manuscript we describe specific techniques for the quantification and characterization of atherosclerotic lesions in the murine aortic sinus and ascending aorta. The advantage of this procedure is that it provides an accurate measurement of the cross-sectional area and total volume of the lesion, which can be used to compare atherosclerotic progression across different treatment groups. This is possible through the use of the valve leaflets as an anatomical landmark, together with careful adjustment of the sectioning angle. We also describe basic staining methods that can be used to begin to characterize atherosclerotic progression. These can be further modified to investigate antigens of specific interest to the researcher. The described techniques are generally applicable to a wide variety of existing and newly created dietary and genetically-induced models of atherogenesis.

We describe procedures to quantify and characterize atherosclerotic lesions in mouse models using precision sectioning of the aortic sinus and ascending aorta combined with histochemical and immunohistochemical analysis.

Atherosclerosis is a disease of the large arteries and a major underlying cause of myocardial infarction and stroke. Several different mouse models have ...

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 ...

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 ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

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

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

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

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.