We thank Mrs. Angela Freund for the excellent technical assistance in sectioning the plastic embedded stents. We thank also Mrs. Roya Soltan and Mrs. Angela Freund for the professional help with immunohistochemistry staining.

1. 	Stent Preparing and Implantation
<ol>
 <li>The stent-struts (Fort Wayne Metals, Castlebar, Ireland) were braided and then cut to the desired size at the Institute for Textile Technology and Mechanical Engineering, RWTH Aachen University in Germany (Figure 1A).</li>
 <li>Before implantation, the stents must be transferred into a 2 cm silicon tube, using forceps, and placed 2 mm at one terminal end, referred front end (Figure 1A).</li>
 <li>The front end should be cut obliquely, to ensure a sharp tip for implantation.</li>
 <li>Before implantation, the stent should be abundantly watered, to ensure slippage.</li>
</ol>
2. 	Stent Implantation
<ol>
 <li>10-12 weeks old male C57Bl/6 wild type mice, 25-27 g are anesthetized using intraperitoneal injection of 100 mg/kg ketamine and 10 mg/kg xylazine. Proper anesthetization is confirmed prior to surgery by the lack of reflexes and beard movement. To prevent dryness while under anesthesia, the mouse eyes are covered by a film of bepanthene cream.</li>
 <li>After shaving and proper disinfection of the ventral neck area, a small median incision of 1 cm is performed under a stereomicroscope, using scissors. After separating the 2 fatty bodies with sterile curved forceps, the left common carotid artery can be seen pulsing along with the trachea.</li>
 <li>1 cm of the left common carotid artery and the bifurcation should be free prepared. 1 knot using a 5/0 silk thread will be bound around the left common carotid artery, 2 knots using 7/0 silk threads will be bound around the left external carotid artery, and 1 knot using a 7/0 silk thread will be bound around the internal carotid artery (Figure 1B).</li>
 <li>The blood flow is then interrupted by binding the knots on the internal carotid artery and the proximal external carotid artery firmly, as well as by pulling the knot surrounding the common carotid artery. The vessel should be fixed in a way that the common and external carotid artery are in a straight line.</li>
 <li>A small incision at the external carotid artery is performed, near the proximal knot, using a Vannas scissor. The silicon tube containing the stent is introduced into the external carotid artery, with the sharp end in front, using a guide-wire. After the stent reaches the desired position, the silicon tube is pulled back over the guide-wire and allows the shape-memory expansion of the stent (Figure 1B).</li>
 <li>The distal knot on the external carotid artery is bind tightly to close the site of incision and the knots at the internal and common carotid artery are removed, thereby restoring the blood flow.</li>
 <li>The skin incision is closed using 3-4 Michel suture clips and a Michel forcep. The mouse is placed under the red light until full recovery. An analgesic treatment is not necessary.</li>
 <li>The plaque can be analyzed after 1-3 weeks. To study the re-endothelialization, an earlier end-time point is necessary (3-4 days). We observed in our model of stent implantation that 4 weeks after this surgical intervention, especially by the use of specific coatings to biofunctionalize the miniaturized stents, neoangiogenesis occurs in approximately 30% of specimen. This is a hind for remodeling and regenerative processes, with different mechanisms and representing another pathological problem. To concentrate on neointima formation, in-stent stenosis and/or analysis of mechanisms underlying these side-effects after stent implantation an end-time point of 3 weeks would be beneficial not to mix up with the regenerative effects induced by the onset of neoangiogenesis.</li>
</ol>
3. 	Analysis of Plaque Formation 
<ol>
 <li>At the end-time point, the animals are anesthetized using intraperitoneal injection of 100 mg/kg ketamine and 10 mg/kg xylazine. Proper anesthetization is confirmed prior to surgery by the lack of reflexes and beard movement.</li>
 <li>The animals are killed by intracardial exsanguination. The serum blood is collected for further analysis.</li>
 <li>After opening the thoracic cavity and PBS washing via intracardial punction, a body-perfusion with 4% paraformaldehyde (PFA) solution is performed for 5 min. The left carotid artery containing the stent is dissected, directly placed in a 4% PFA solution and at least 16 hr later embbeded in plastic.</li>
 <li>50 &mu;m thick sections are performed from plastic-embedded samples using a diamond band saw.</li>
 <li>To measure the plaque size, Giemsa staining is performed.</li>
 <li>To analyze the rate of re-endothelialization within the stented area of the vessel, immunohistochemistry for von Willebrand factor (vWF) is performed.</li>
</ol>

<ol>
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Cardiol. 47, 13-18 (2006).</li><li>Farb, 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=Pathology+of+acute+and+chronic+coronary+stenting+in+humans.">Pathology of acute and chronic coronary stenting in humans.</a> Circulation. 99, 44-52 (1999).</li><li>Weber, C., Noels, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atherosclerosis:+current+pathogenesis+and+therapeutic+options.">Atherosclerosis: current pathogenesis and therapeutic options.</a> Nat. Med. 17, 1410-1422 (2011).</li><li>Lenzen, M. 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=Management+and+outcome+of+patients+with+established+coronary+artery+disease:+the+Euro+Heart+Survey+on+coronary+revascularization.">Management and outcome of patients with established coronary artery disease: the Euro Heart Survey on coronary revascularization.</a> Eur. Heart J. 26, 1169-1179 (2005).</li><li>Babapulle, M. N., 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+hierarchical+Bayesian+meta-analysis+of+randomised+clinical+trials+of+drug-eluting+stents.">A hierarchical Bayesian meta-analysis of randomised clinical trials of drug-eluting stents.</a> Lancet. 364, 583-591 (2004).</li><li>Wiviott, S. D., 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=Intensive+oral+antiplatelet+therapy+for+reduction+of+ischaemic+events+including+stent+thrombosis+in+patients+with+acute+coronary+syndromes+treated+with+percutaneous+coronary+intervention+and+stenting+in+the+TRITON-TIMI+38+trial:+a+subanalysis+of+a+randomised+trial.">Intensive oral antiplatelet therapy for reduction of ischaemic events including stent thrombosis in patients with acute coronary syndromes treated with percutaneous coronary intervention and stenting in the TRITON-TIMI 38 trial: a subanalysis of a randomised trial.</a> Lancet. 371, 1353-1363 (2008).</li><li>van Werkum, J. W., 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=Predictors+of+coronary+stent+thrombosis:+the+Dutch+Stent+Thrombosis+Registry.">Predictors of coronary stent thrombosis: the Dutch Stent Thrombosis Registry.</a> J. Am. Coll. Cardiol. 53, 1399-1409 (2009).</li><li>Finn, A. V., 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=Vascular+responses+to+drug+eluting+stents:+importance+of+delayed+healing.">Vascular responses to drug eluting stents: importance of delayed healing.</a> Arterioscler. Thromb Vasc. Biol. 27, 1500-1510 (2007).</li><li>Joner, M., 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=Pathology+of+drug-eluting+stents+in+humans:+delayed+healing+and+late+thrombotic+risk.">Pathology of drug-eluting stents in humans: delayed healing and late thrombotic risk.</a> J. Am. Coll. Cardiol. 48, 193-202 (2006).</li><li>Cines, D. B., 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=Endothelial+cells+in+physiology+and+in+the+pathophysiology+of+vascular+disorders.">Endothelial cells in physiology and in the pathophysiology of vascular disorders.</a> Blood. 91, 3527-3561 (1998).</li><li>Hristov, M., Weber, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+progenitor+cells:+characterization,+pathophysiology,+and+possible+clinical+relevance.">Endothelial progenitor cells: characterization, pathophysiology, and possible clinical relevance.</a> J. Cell Mol. Med. 8, 498-508 (2004).</li><li>Rabelink, T. 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=Endothelial+progenitor+cells:+more+than+an+inflammatory+response?.">Endothelial progenitor cells: more than an inflammatory response?.</a> Arterioscler. Thromb. Vasc. Biol. 24, 834-838 (2004).</li><li>Schwartz, R. 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=Preclinical+evaluation+of+drug-eluting+stents+for+peripheral+applications:+recommendations+from+an+expert+consensus+group.">Preclinical evaluation of drug-eluting stents for peripheral applications: recommendations from an expert consensus group.</a> Circulation. 110, 2498-2505 (2004).</li><li>Schwartz, R. 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=Differential+neointimal+response+to+coronary+artery+injury+in+pigs+and+dogs.+Implications+for+restenosis+models.">Differential neointimal response to coronary artery injury in pigs and dogs. Implications for restenosis models.</a> Arterioscler. Thromb. 14, 395-400 (1994).</li><li>Schwartz, R. 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=Restenosis+and+the+proportional+neointimal+response+to+coronary+artery+injury:+results+in+a+porcine+model.">Restenosis and the proportional neointimal response to coronary artery injury: results in a porcine model.</a> J. Am. Coll. Cardiol. 19, 267-274 (1992).</li><li>Ali, Z. 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, 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, 359-366 (2010).</li><li>Costa, F., 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=Covalent+immobilization+of+antimicrobial+peptides+(AMPs)+onto+biomaterial+surfaces.">Covalent immobilization of antimicrobial peptides (AMPs) onto biomaterial surfaces.</a> Acta Biomaterialia. 7, 1431-1440 (2011).</li><li>Soehnlein, O., 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=Neutrophil-derived+cathelicidin+protects+from+neointimal+hyperplasia.">Neutrophil-derived cathelicidin protects from neointimal hyperplasia.</a> Science Translational Medicine. 3, 103ra198 (2011).</li></ol>

No conflicts of interest declared.

To reduce the risk of in-stent thrombosis and restenosis and to sustain the development of new coatings for drug-eluting stents, an easy, simple and accessible method of stent implantation in an animal model is needed. Mice deliver the ideal system to study the complex mechanisms of arterial remodeling after stent implantation and the efficiency of such drugs. Existing models for in-stent restenosis in mouse are difficult, require high surgical skills and imply high risks of complications as bleeding or paralysis17-19. For example, in the model of the stent- implantation into thoracic aorta of a donor mouse after balloon-dilatation of the vessel and then transplantation of the stented segment into carotid artery of a recipient mouse17, the study of the patho-mechanisms is not influenced only by recipient reaction to donor material, but also by the massive damaging of vasa vasorum and adventitia. Implantation of a stainless steel stent directly into abdominal aorta after balloon-dilatation19 is followed by a high mortality rate (35%) because of hind leg paralysis after thrombosis or bleeding from abdominal aorta on site of arteriotomy. Implantation of a spiral-shaped self-expanding nitinol-stent into abdominal aorta via femoral artery18 needs high surgical skills, mostly due to blindly directing the stent along the branching from femoral artery to aorta to place the stent at the right position. This procedure is followed by a high risk of damaging the femoral nerve, therefore paralysis of the hind leg. Compared with these procedures, our model of stent implantation in mouse does not need high surgical skills.
Our model offers a simple, easy and efficient method to analyze the effects of different drug-coatings on arterial remodeling, the placing of the stent is made under sight, and there are no risks of damaging nerves or other structures. The complex molecular mechanisms can be investigated easier in our model of mouse carotid artery stenting, not only by direct accessibility of the vessel, but also due to the existence of different knock-out mice strains.
As one limitation, comparing with the clinical procedure, our model uses healthy mice/arteries and doesn't perform stenting on pre-existing plaques (not in-stent restenosis, but in-stent stenosis). We also don't perform balloon-dilatation prior to stent-implantation. However, due to the massive damage of the vessel wall in both models, the reparatory processes are similar. Unfortunately, due to the metal-derived artifacts, an in vivo monitoring of the neointimal growth is not possible by existing imaging methods as ultrasound or computer-tomography. Another limiting factor is the thin sectioning of metal-based stents, which requires some expertise in the metal processing.
Using this method, we were able to show, that neutrophil-instructing biofunctionalized miniaturized nitinol-stents coated with LL-37 reduce in-stent restenosis, providing a novel concept to promote vascular healing after interventional therapy21.
Despite these limitations, this model seems to be, until now, the most suitable system, thereby money- and time-saving, to investigate new drug-coatings for stents and their effects on the molecular events during arterial remodeling. Moreover, this model can be easily adapted to the hamster, which is more similar to the human, so that every therapeutical hypothesis can be verified before applying to larger animals or human to avoid unpleasant and unexpected effects.

Cardiovascular diseases caused by the progression of atherosclerosis are the leading cause of death in the industrialized nations. Atherosclerosis is a focal, inflammatory fibro-proliferative response of the vascular wall to endothelial injury1, resulting in the formation of an extended plaque into the lumen of the vessel, affecting the blood flow through coronary arteries. Over 75% of myocardial infarctions result from the rupture of the thin fibrous cap of the inflamed plaque2. Since this complication can be fatal, a percutaneous transluminal (coronary) angioplasty (PTCA) with stent implantation became the first-choice therapy in the current medical practice. The method allows the dilatation of the narrowed coronary artery and thus the restoration of blood flow. Simultaneously, it causes an extent injury to the endothelium and vessel wall3. However, the long-term effect of this therapy is limited by an excessive arterial remodeling and restenosis4.
By employment of stents, the PTCA became more effective in the treatment of complicated lesions, allowing revascularization after an acute vessel closure5. This method decreases the incidence of in-stent restenosis to less than 10%6. Beside these benefits, this first-choice therapy for coronary revascularization bears also the life-threatening risks of in-stent thrombosis and restenosis.
In-stent thrombosis is caused by a de-endothelialization of the vessel, followed by a massive adhesion of platelets and fibrin to the injured site. 26% of patients suffer from in-stent thrombosis and 63% die of myocardial infarction7. Restenosis refers to the process of wound healing after mechanical injury to the vessel wall, involving neointimal hyperplasia (migration and proliferation of vascular smooth muscle cells (VSMC), deposition of extracellular matrix (ECM), and remodeling of the vessel. Often, an invasive re-intervention becomes necessary to dilatate severely narrowed atherosclerotic vessels due to in-stent thrombosis and restenosis.
To prevent in-stent thrombosis, a long-term treatment with anti-thrombotic drugs is necessary8. To prevent restenosis, new generation of drug-eluting stents elute anti-proliferative agents such as immunosuppressive drugs (e.g. sirolimus, everolimus, zotarolimus) and anti-cancer drugs (e.g. paclitaxel) from a polymer coating for several months9,10. Although these drugs decrease the neointima formation and restenosis, they maintain a high risk of in-stent thrombosis by inhibiting the re-endothelialization.
After arterial injury, the maintenance of the endothelial compartment is essential to prevent thrombotic complications. Under physiological conditions, the human endothelium shows a small turnover rate11. Under pathological conditions, however, the endothelial integrity is impaired, so that a rapid recovery by surrounding mature endothelial cells and circulating endothelial progenitor cells (EPCs) is required12,13.
The study of these complex molecular mechanisms in larger animals14-16 or in mouse aortic artery is a very difficult procedure, offering limited data17-19. To test the efficiency of novel stent-coatings to reduce in-stent thrombosis and restenosis new models are imperative.
Nitinol represents the ideal platform for stents because of its' high elasticity, shape-memory effect and good tolerance in patients, being successfully used as bare-metal stents in clinical use. This alloy made it possible to create a miniaturized stent with an external diameter of 500 &mu;m, which can be coated20 and implanted into the carotid artery of mice. The development of a miniaturized nitinol stent for mouse carotid artery, allows the study of precise molecular mechanisms induced by stent implantation and offers the possibility to test quickly and efficiently the effects of different drug-coatings to prevent restenosis. Moreover, the existence of different knock-out mice strains represents a huge advantage in clarifying the role of different molecules involved in neointima growth and in-stent thrombosis.

<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments (optional)</td>
		</tr>
		<tr>
			<td>nitinol-stents (self-made from nitinol-struts)</td>
			<td>Fort Wayne Metals, Castlebar, Ireland NiTi#1, superelastic, straight annealed, light oxide, diameter 500 &mu;m</td>
			<td>custom-made product</td>
			<td>Institute for Textile Technology and Mechanical Engineering</td>
		</tr>
		<tr>
			<td>silicon tube</td>
			<td>IFK Isofluor, Germany</td>
			<td>custom-made product</td>
			<td>diameter 500 &mu;m, section thickness 100 &mu;m, polytetrafluorethylene catheter</td>
		</tr>
		<tr>
			<td>stereomicroscope</td>
			<td>Olympus</td>
			<td>SZ/X9</td>
			<td>&nbsp;</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>Ketamine 10%</td>
			<td>CEVA, Germany</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Xylazine 2%</td>
			<td>Medistar, Germany</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Bepanthene</td>
			<td>Bayer, Germany</td>
			<td>&nbsp;</td>
			<td>&nbsp;</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>&nbsp;</td>
		</tr>
		<tr>
			<td>5/0 Silk</td>
			<td>Seraflex</td>
			<td>IC 108000</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>7/0 Silk</td>
			<td>Seraflex</td>
			<td>IC 1005171Z</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>guide-wire</td>
			<td>Abbott Vascular</td>
			<td>1001782-HC</td>
			<td>0.014-inch angioplastie guide-wire</td>
		</tr>
		<tr>
			<td>Michel suture clips</td>
			<td>Aesculap, Germany</td>
			<td>BN507R</td>
			<td>7.5 x 1.75 mm</td>
		</tr>
		<tr>
			<td>Michel Forcep</td>
			<td>Aesculap, Germany</td>
			<td>BN730R</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

a murine model of stent implantation in the carotid artery for the study of restenosis

Despite the considerable progress made in the stent development in the last decades, cardiovascular diseases remain the main cause of death in western countries. Beside the benefits offered by the development of different drug-eluting stents, the coronary revascularization bears also the life-threatening risks of in-stent thrombosis and restenosis. Research on new therapeutic strategies is impaired by the lack of appropriate methods to study stent implantation and restenosis processes. Here, we describe a rapid and accessible procedure of stent implantation in mouse carotid artery, which offers the possibility to study in a convenient way the molecular mechanisms of vessel remodeling and the effects of different drug coatings.

<ol>
	<li>The implantation of a miniaturized nitinol stent into the left carotid artery of mice takes 25-30 min and shows a mortality rate of 10% mostly due to the damage of the vessel during the intervention. A better survival rate is observed in mice having a weight more than 25 g at the time of stent implantation (mortality rate of 5%). Therefore, we chose for the implantation mice with a weight between 25-27 g. After surgery, the mice recover from anesthesia within 2-5 min and no physical impairments, like e.g. paralysis, are observed. Micro-Computer Tomography (Micro-CT) imaging performed one week after stent implantation showed that the stents are not dislocated by blood flow (Figure 1C). Unfortunately, analysis of neointima formation in these images is not possible due to the metal-derived artifacts (Figure 1D, 1E).</li>
	<li>We didn't observe any vessel or endothelial damages of unstented area of the vessel, immediately below the stent, as detectable by histological (Figure 2A) and by specific staining for endothelium (Figure 2B, anti-mouse CD31 antibody). For a better overview, the section was scanned using a two-photon laser scanning microscopy (Figure 2B, 2C).</li>
	<li>In the stented vessel, a permanent dilatation of 15% is detected (ratio stent:artery, 1,15:1) by mice with a weight between 25-27 g. Neointima formation and thrombus-formation can be analyzed by classical histological stainings (e.g. Hematoxilin-eosin, Giemsa, Movat, Toluidin Blue, Masson-Trichrom-Goldner, Figure 3A, 3B). Since the lamina externa and interna are not visible anymore, plaque size was calculated as the difference of the external and the luminal areas (mean plaque area: 234566 &plusmn; 3315 &mu;m2, mean luminal area: 12036 &plusmn; 2662 &mu;m2). External circumference was also measured (mean: 1799 &plusmn; 14 &mu;m). For analysis of the cellular composition, the sections need to be deplastified and stained with specific markers. For the re-endothelialization, we used a Cy3-conjugated anti-CD31 antibody and for smooth muscle cell proliferation a FITC-conjugated anti-SMA antibody (Figure 3C). Re-endothelialization was calculated as percentage of CD-31 positive stained to the total luminal surface (mean: 23.07 &plusmn; 3.14 %) one week after stent implantation.</li>
</ol>
Of course, an unlimited number of specific staining is possible, depending on each laboratories' experience. Analysis of myosin heavy chain, for a better characterization of SMCs, but also analysis of infiltrated cells (monocytes, lymphocytes), or stainings for different inflammatory cytokines can also be performed, depending on the aim of the study.
<img alt="Figure 1" fo:content-width="6in" fo:src="/files/ftp_upload/50233/50233fig1highres.jpg" src="/files/ftp_upload/50233/50233fig1.jpg" /> 
Figure 1. Schematic overview of the surgical procedure (A). The blood flow is interrupted by binding the knots on the internal carotid artery and the proximal external carotid artery firmly, as well as by pulling the knot surrounding the common carotid artery. The silicon tube containing the stent is introduced into the external carotid artery through a small incision at the external carotid artery. After the stent reaches the desired position, the silicon tube is pulled back over the guide-wire and allows the shape-memory expansion of the stent. Micro-CT images showing the stent position one week after surgical implantation (B). Due to the material-derived artifacts, an analysis of the neointima growth is not possible (C,D).
<img alt="Figure 2" fo:content-width="6in" fo:src="/files/ftp_upload/50233/50233fig2highres.jpg" src="/files/ftp_upload/50233/50233fig2.jpg" /> 
Figure 2. Unstented area of the vessel is not affected by the surgical procedure, as shown by Toluidin Blue (A) and endothelial-specific CD31 staining (B, C).
<img alt="Figure 3" fo:content-width="6in" fo:src="/files/ftp_upload/50233/50233fig3highres.jpg" src="/files/ftp_upload/50233/50233fig3.jpg" /> 
Figure 3. Analysis of the plaque can be performed by classical histological stainings (e.g. Masson-Trichrom-Goldner) (A). The organized thrombus can be detected by black-stained fibrin depositions inside the neointima, in some cases a complete occlusion of the vessel is observed (B). Re-endothelialization (Cy3, red) or smooth muscle cell proliferation (FITC, green) was detected by double immunofluorescence staining using specific markers. Counterstaining was performed with 4',6-diamidino-2-phenylindole (DAPI, blue) (C). We noticed a completed re-endothelialization of the stent struts (left, double arrow) compared to an incompleted luminal re-endothelialization (right, single arrow).

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A Murine Model of Stent Implantation in the Carotid Artery for the Study of Restenosis

A model of stent implantation in mouse carotid artery is described. Compared to other similar methods, this procedure is very rapid, simple and accessible, offering the possibility to study in a convenient way the vascular wall reaction to different drug-eluting stents and the molecular mechanisms of restenosis.

Despite  the considerable progress made in the stent development in the last decades,  cardiovascular diseases remain the main cause of death in western ...

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Research

JoVE Journal

Medicine

25.3K vistas.  RWTH Aachen University. Se describe un modelo de implantación de un stent en la arteria carótida del ratón. En comparación con otros métodos similares, este procedimiento es muy rápido, simple y accesible, que ofrece la posibilidad de estudiar de una manera conveniente la reacción de la pared vascular a diferentes stents liberadores de fármacos y los mecanismos moleculares de la restenosis.

Video: A Murine Model of Stent Implantation in the Carotid Artery for the Study of Restenosis

Biomarkers in an Animal Model for Revealing Neural, Hematologic, and Behavioral Correlates of PTSD

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for objective diagnosis but also for the evaluation of therapeutic efficacy and resilience to trauma. Ongoing research is directed at identifying molecular biomarkers for PTSD, including traumatic stress induced proteins, transcriptomes, genomic variances and genetic modulators, using biologic samples from subjects' blood, saliva, urine, and postmortem brain tissues. However, the correlation of these biomarker molecules in peripheral or postmortem samples to altered brain functions associated with psychiatric symptoms in PTSD remains unresolved. Here, we present an animal model of PTSD in which both peripheral blood and central brain biomarkers, as well as behavioral phenotype, can be collected and measured, thus providing the needed correlation of the central biomarkers of PTSD, which are mechanistic and pathognomonic but cannot be collected from people, with the peripheral biomarkers and behavioral phenotypes, which can.
Our animal model of PTSD employs restraint and tail shocks repeated for three continuous days - the inescapable tail-shock model (ITS) in rats. This ITS model mimics the pathophysiology of PTSD 17, 7, 4, 10. We and others have verified that the ITS model induces behavioral and neurobiological alterations similar to those found in PTSD subjects 17, 7, 10, 9. Specifically, these stressed rats exhibit (1) a delayed and exaggerated startle response appearing several days after stressor cessation, which given the compressed time scale of the rat's life compared to a humans, corresponds to the one to three months delay of symptoms in PTSD patients (DSM-IV-TR PTSD Criterian D/E 13), (2) enhanced plasma corticosterone (CORT) for several days, indicating compromise of the hypothalamopituitary axis (HPA), and (3) retarded body weight gain after stressor cessation, indicating dysfunction of metabolic regulation.
The experimental paradigms employed for this model are: (1) a learned helplessness paradigm in the rat assayed by measurement of acoustic startle response (ASR) and a charting of body mass; (2) microdissection of the rat brain into regions and nuclei; (3) enzyme-linked immunosorbent assay (ELISA) for blood levels of CORT; (4) a gene expression microarray plus related bioinformatics tools 18. This microarray, dubbed rMNChip, focuses on mitochondrial and mitochondria-related nuclear genes in the rat so as to specifically address the neuronal bioenergetics hypothesized to be involved in PTSD.

We describe a rat model of post traumatic stress disorder (PTSD) that reveals the persistent alterations in neuroendocrine function and the delayed long-term, exaggerated fear response, characteristic of PTSD patients. The animal model and methods described here are useful for correlating biomarkers in brain nuclei, which are mechanistic but cannot be measured in patients, with biomarkers in peripheral white blood cells, which can.

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for ...

Human Internal Mammary Artery (IMA) Transplantation and Stenting: A Human Model to Study the Development of In-Stent Restenosis

Preclinical in vivo research models to investigate pathobiological and pathophysiological processes in the development of intimal hyperplasia after vessel stenting are crucial for translational approaches1,2.
The commonly used animal models include mice, rats, rabbits, and pigs3-5. However, the translation of these models into clinical settings remains difficult, since those biological processes are already studied in animal vessels but never performed before in human research models6,7. In this video we demonstrate a new humanized model to overcome this translational gap. The shown procedure is reproducible, easy, and fast to perform and is suitable to study the development of intimal hyperplasia and the applicability of diverse stents.
This video shows how to perform the stent technique in human vessels followed by transplantation into immunodeficient rats, and identifies the origin of proliferating cells as human.

Human Internal Mammary Artery IMA Transplantation and Stenting: A Human Model to Study the Development of In-Stent Restenosis

This video shows a model to study the development of intimal hyperplasia after stent deployment using a human vessel (IMA) in an immunodeficient rat model.

Preclinical in vivo research models to investigate pathobiological and pathophysiological processes in the development of intimal hyperplasia after vessel ...

Training a Sophisticated Microsurgical Technique: Interposition of External Jugular Vein Graft in the Common Carotid Artery in Rats

Neointimal hyperplasia is one the primary causes of stenosis in arterialized veins that are of great importance in arterial coronary bypass surgery, in peripheral arterial bypass surgery as well as in arteriovenous fistulas.1-5 The experimental procedure of vein graft interposition in the common carotid artery by using the cuff-technique has been applied in several research projects to examine the aetiology of neointimal hyperplasia and therapeutic options to address it. 6-8 The cuff prevents vessel anastomotic remodeling and induces turbulence within the graft and thereby the development of neointimal hyperplasia.
Using the superior caval vein graft is an established small-animal model for venous arterialization experiment.9-11 This current protocol refers to an established jugular vein graft interposition technique first described by Zou et al., 9 as well as others.12-14 Nevertheless, these cited small animal protocols are complicated.
To simplify the procedure and to minimize the number of experimental animals needed, a detailed operation protocol by video training is presented. This video should help the novice surgeon to learn both the cuff-technique and the vein graft interposition. Hereby, the right external jugular vein was grafted in cuff-technique in the common carotid artery of 21 female Sprague Dawley rats categorized in three equal groups that were sacrificed on day 21, 42 and 84, respectively. Notably, no donor animals were needed, because auto-transplantations were performed. The survival rate was 100 % at the time point of sacrifice. In addition, the graft patency rate was 60 % for the first 10 operated animals and 82 % for the remaining 11 animals. The blood flow at the time of sacrifice was 8&plusmn;3 ml/min. In conclusion, this surgical protocol considerably simplifies, optimizes and standardizes this complicated procedure. It gives novice surgeons easy, step-by-step instruction, explaining possible pitfalls, thereby helping them to gain expertise fast and avoid useless sacrifice of experimental animals.

Neointimal hyperplasia is the primary cause of stenosis in arterialized veins. We propose a new protocol whereby the right external jugular vein is grafted using the cuff technique in the common carotid artery of Sprague Dawley rats. The survival rate was 100 % at the time point of sacrifice.

Neointimal hyperplasia is one the primary causes of stenosis in arterialized veins that are of great importance in arterial coronary bypass surgery, in ...

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

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

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

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

Carotid Artery Infusions for Pharmacokinetic and Pharmacodynamic Analysis of Taxanes in Mice

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study model. As the use of mouse models are often a vital step in preclinical drug discovery and drug development1-8, it is necessary to design a system to introduce drugs into mice in a uniform, reproducible manner. Ideally, the system should permit the collection of blood samples at regular intervals over a set time course. The ability to measure drug concentrations by mass-spectrometry, has allowed investigators to follow the changes in plasma drug levels over time in individual mice1, 9, 10. In this study, paclitaxel was introduced into transgenic mice as a continuous arterial infusion over three hours, while blood samples were simultaneously taken by retro-orbital bleeds at set time points. Carotid artery infusions are a potential alternative to jugular vein infusions, when factors such as mammary tumors or other obstructions make jugular infusions impractical. Using this technique, paclitaxel concentrations in plasma and tissue achieved similar levels as compared to jugular infusion. In this tutorial, we will demonstrate how to successfully catheterize the carotid artery by preparing an optimized catheter for the individual mouse model, then show how to insert and secure the catheter into the mouse carotid artery, thread the end of the catheter out through the back of the mouse&#8217;s neck, and hook the mouse to a pump to deliver a controlled rate of drug influx. Multiple low volume retro-orbital bleeds allow for analysis of plasma drug concentrations over time.

This method was developed with the goal of delivering a steady drug solution via the carotid artery, to assess the pharmacokinetics of novel drugs in mouse models.

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study ...

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 ...

Performing Permanent Distal Middle Cerebral with Common Carotid Artery Occlusion in Aged Rats to Study Cortical Ischemia with Sustained Disability

Stroke typically occurs in elderly people with a range of comorbidities including carotid (or other arterial) atherosclerosis, high blood pressure, obesity and diabetes. Accordingly, when evaluating therapies for stroke in animals, it is important to select a model with excellent face validity. Ischemic stroke accounts for 80% of all strokes, and the majority of these occur in the territory of the middle cerebral artery (MCA), often inducing infarcts that affect the sensorimotor cortex, causing persistent plegia or paresis on the contralateral side of the body. We demonstrate in this video a method for producing ischemic stroke in elderly rats, which causes sustained sensorimotor disability and substantial cortical infarcts. Specifically, we induce permanent distal middle cerebral artery occlusion (MCAO) in elderly female rats by using diathermy forceps to occlude a short segment of this artery. The carotid artery on the ipsilateral side to the lesion was then permanently occluded and the contralateral carotid artery was transiently occluded for 60 min. We measure the infarct size using structural T2-weighted magnetic resonance imaging (MRI) at 24 hr and 8 weeks after stroke. In this study, the mean infarct volume was 4.5% &#177; 2.0% (standard deviation) of the ipsilateral hemisphere at 24 hr (corrected for brain swelling using Gerriet&#8217;s equation, n = 5). This model is feasible and clinically relevant as it permits the induction of sustained sensorimotor deficits, which is important for the elucidation of pathophysiological mechanisms and novel treatments.

Here we present a protocol to produce permanent distal middle cerebral artery occlusion in elderly female rats with simultaneous occlusion of the carotid arteries to generate large cortical infarcts and sustained deficits. We show confirmation of the lesion size using structural MRI at 24 hr and 8 weeks after stroke.

Stroke typically occurs in elderly people with a range of comorbidities including carotid (or other arterial) atherosclerosis, high blood pressure, ...

In Vivo Gene Transfer to the Rabbit Common Carotid Artery Endothelium

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

In Vivo Gene Transfer to the Rabbit Common Carotid Artery Endothelium

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

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

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

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

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

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