This work was supported by a Wisconsin Partnership Program New Investigator Award (ID 2832), a National Heart, Lung, Blood Institute R01 Grant (HL-068673) and a T32 training Grant (HL-110853). We thank Dr. Melina Kibbe&#8217;s group at Northwestern University for providing helpful information.

NOTE: Ethics Statement: All procedures conform to the Guide for the Care and Use of Laboratory Animals (National Academies Press, 8th edition, 2011),&#160;&#160;and protocols approved by the Institutional Animal Care and Use Committee at the University of Wisconsin. All surgeries were performed under isoflurane anesthesia (through inhaling, flow rate 2 ml/min), and all efforts were made to minimize suffering. Animals were euthanized in a chamber gradually filled with CO2.
1. Induction of General Anesthesia and Skin Incision
<ol>
	<li>Use male C57BL/6 mice with an age of 12&#8211;16 weeks and weight of 23&#8211;28 g. Anesthetize the animal by isoflurane through a closed-circuit anesthetic apparatus. Confirm proper anesthetization by pinching its toe. Apply some artificial tears ointment on eyes to prevent dryness.</li>
	<li>Place the mouse in a supine position with the left hind limb slightly abducted and the knee joint slightly flexed. Shave the hair around the left upper thigh region and disinfect the skin with&#160;Chlorhexidine, applied three times using sterile cotton tipped applicators. Use a hot bead sterilizer to keep the instruments sterilized during the surgery.</li>
	<li>Place a sterilized gauze drape over the left leg. Make a 1.5 cm straight, longitudinal skin incision on the left medial thigh from the knee joint&#160;to the groin (Figure 1A).</li>
</ol>
2. Exposure of Femoral Artery and Branches
NOTE: Figure 1B shows the gross vasculature anatomy in the upper thigh. The superficial femoral artery (SFA) can be observed through a dissecting microscope when the skin incision is properly made along the midline of the medial thigh.
<ol>
	<li>Open up the thin fascia overlying the SFA with fine tweezers and then loop the SFA with 9-0 nylon suture. 
	NOTE: In this process, the femoral nerve is easily put aside, but the adjacent femoral vein requires close attention because its wall is so thin that it easily perforates and bleeds. The key technique is to focus on the artery itself; as long as the dissection is carried out close to the artery, any accidental vein damage is avoidable.</li>
	<li>Once the SFA is looped, hold the suture with mosquito forceps and pull it slightly downward. This maneuver makes further dissection easier, but to avoid damaging the artery do not pull too forcefully.</li>
	<li>Continue dissecting the artery proximally to expose and loop the common femoral artery (CFA) with 9-0 nylon suture. 
	NOTE: Usually, looping the CFA at a point distal to the epigastric artery (Figure 1B) is enough for wire insertion. Hold the CFA suture loop with mosquito forceps in the same way.</li>
	<li>Pull the CFA suture loop upward and the SFA suture loop downward to uncover the bifurcation of SFA and deep femoral artery (DFA). The DFA, the major branch of the CFA, goes toward the thigh muscle (Figure 1B). 
	NOTE: The wire will be inserted from the DFA later.</li>
	<li>Loop the DFA with extra caution because the adjacent vein is tangled beneath the artery. In case of bleeding from the vein, a 30- to 60 sec&#160;simple compression with gauze is usually sufficient to produce hemostasis.</li>
	<li>Once the DFA is looped with 9-0 nylon suture, make sure all three suture loops are at their correct positions (Figure 2A).</li>
	<li>Proceed with the dissection of the DFA distally to ligate the artery at a point as far as possible from the bifurcation. Cut extra suture but leave a certain length so that the two ends can be held with mosquito forceps, which stabilize the DFA making the wire insertion easier.</li>
</ol>
3. Creating Wire Injury
<ol>
	<li>Insert Wire via DFA
	<ol>
		<li>Strain the CFA, SFA, and DFA sutures to establish a temporary hemostat.</li>
		<li>Make a small hole in the DFA with microscissors. Insert the tips of the curved microtweezers to dilate the hole large enough for a wire with a 0.015 inch diameter (Figure 2B demonstrates the size difference between the artery and the wire).
		<ol>
			<li>Before making a hole in the DFA, loosen the CFA suture loop and gently clamp/squeeze the DFA several times with dull-tipped microtweezers. This maneuver expands the arterial diameter remarkably to ease the wire insertion. Moistening the wire thoroughly with PBS is also important for a smooth insertion.</li>
			<li>When making a hole in the DFA, do not crop the arterial wall, but make a small v-shaped nip and pinch the flap with sharp-tipped microtweezers for manipulation.</li>
		</ol>
		</li>
		<li>Insert the wire through the adequately-dilated hole on the DFA. Gently advance the wire toward the iliac artery while loosening the suture loop around the CFA. 5&#8211;8 mm is enough to cover all the CFA length in most cases (Figure 2C). Do not insert the wire forcefully as an arterial perforation usually results in loss of the mouse. Shortening the wire to about 15-mm in length helps with handling.</li>
	</ol>
	</li>
	<li>Leave Wire inside for 1 Min
	<ol>
		<li>After inserting the wire to the optimal length, leave it for 1 min to overstretch the arterial wall. Moisten the wire and arteries with PBS to prevent drying. Another 9-0 nylon suture can be placed around the DFA proximal to the wire-insertion point during this waiting time. This suture will be used later to close the hole on the DFA following removal of the wire.</li>
	</ol>
	</li>
	<li>Retrieve Wire and Resume the Blood Flow
	<ol>
		<li>Hold the wire with vessel cannulation forceps, and pull it back gently; the wire should be retrieved smoothly. To overcome any resistance, usually caused by adherence of the wire to the arterial wall, pulling back the wire with gentle rotation is helpful. Again, a forceful maneuver could result in animal loss.</li>
		<li>After retrieving the wire, ligate the DFA at the point of bifurcation with 9-0 nylon suture. Do not ligate too close to the bifurcation, which would jeopardize the blood outflow to the SFA.</li>
		<li>Loosen the SFA and CFA suture loops to resume the normal blood flow. Note that the CFA is well-expanded by wire insertion (Figure 2D).</li>
	</ol>
	</li>
</ol>
4. Skin Closure and Postoperative Care
<ol>
	<li>Control bleeds. Minor oozing can be stopped by simple compression; some major arterial bleeds will require stitches with a 11-0 nylon suture. These sutured arteries will eventually result in arterial occlusion.</li>
	<li>Close the skin wound with skin staplers (usually 3&#8211;4 staples). Keep the wound area sterilized by&#160;applying Chlorhexidine surgical solution.</li>
	<li>Apply post-surgery treatments. Control the pain of the mouse with subcutaneous injections of buprenorphine. Keep the mouse on the warming pad until it has regained sufficient consciousness to maintain sternal recumbency. House it in a clean cage without company of other animals until fully recovered.</li>
</ol>

<ol>
	<li>Dzau, V. J., Braun-Dullaeus, R. C., Sedding, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular+proliferation+and+atherosclerosis:+new+perspectives+and+therapeutic+strategies.">Vascular proliferation and atherosclerosis: new perspectives and therapeutic strategies.</a> Nat Med. 8 (11), 1249-1256 (2002).</li><li>Hui, D. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intimal+hyperplasia+in+murine+models.">Intimal hyperplasia in murine models.</a> Curr Drug Targets. 9 (3), 251-260 (2008).</li><li>Ferns, G. A., Avades, T. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mechanisms+of+coronary+restenosis:+insights+from+experimental+models.">The mechanisms of coronary restenosis: insights from experimental models.</a> Int J Exp Pathol. 81 (2), 63-88 (2000).</li><li>Sata, 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=A+mouse+model+of+vascular+injury+that+induces+rapid+onset+of+medial+cell+apoptosis+followed+by+reproducible+neointimal+hyperplasia.">A mouse model of vascular injury that induces rapid onset of medial cell apoptosis followed by reproducible neointimal hyperplasia.</a> J Mol Cell Cardiol. 32 (11), 2097-2104 (2000).</li><li>Xu, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+models+of+arteriosclerosis:+from+arterial+injuries+to+vascular+grafts.">Mouse models of arteriosclerosis: from arterial injuries to vascular grafts.</a> Am J Pathol. 165 (1), 1-10 (2004).</li><li>Lv, L., Meng, Q., Ye, M., Wang, P., Xue, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=STAT4+deficiency+protects+against+neointima+formation+following+arterial+injury+in+mice.">STAT4 deficiency protects against neointima formation following arterial injury in mice.</a> J Mol Cell Cardiol. 74C, 284-294 (2014).</li><li>Zhu, B., Kuhel, D. G., Witte, D. P., Hui, D. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apolipoprotein+E+inhibits+neointimal+hyperplasia+after+arterial+injury+in+mice.">Apolipoprotein E inhibits neointimal hyperplasia after arterial injury in mice.</a> Am J Pathol. 157 (6), 1839-1848 (2000).</li><li>Fu, Y., Duru, E. A., Davies, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+metabolic+syndrome+on+the+response+to+arterial+injury.">Effect of metabolic syndrome on the response to arterial injury.</a> J Surg Res. , (2014).</li><li>Kuhel, D. G., Zhu, B., Witte, D. P., Hui, D. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinction+in+genetic+determinants+for+injury-induced+neointimal+hyperplasia+and+diet-induced+atherosclerosis+in+inbred+mice.">Distinction in genetic determinants for injury-induced neointimal hyperplasia and diet-induced atherosclerosis in inbred mice.</a> Arterioscler Thromb Vasc Biol. 22 (6), 955-960 (2002).</li><li>Cooley, B. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+strain+differential+neointimal+response+in+vein+grafts+and+wire-injured+arteries.">Mouse strain differential neointimal response in vein grafts and wire-injured arteries.</a> Circ J. 71 (10), 1649-1652 (2007).</li><li>Ishigami, 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=Deficiency+of+CuZn+superoxide+dismutase+promotes+inflammation+and+alters+medial+structure+following+vascular+injury.">Deficiency of CuZn superoxide dismutase promotes inflammation and alters medial structure following vascular injury.</a> J Atheroscler Thromb. 18 (11), 1009-1017 (2011).</li></ol>

The authors declare no competing financial interests.

The wire injury procedure is applicable to all strains of mice as long as their anatomical structures are similar.4 In this paper, we used male C57BL/6 mice with an age of 12&#8211;16 weeks. As we have learned from our preliminary studies, the femoral arteries of mice younger than 10 weeks are often so small that wire insertion is quite challenging. On the other hand, wire insertion in mice older than 16 weeks is technically easier but tends to result in more variable neointimal hyperplasia. Female mice tend to incur less neointimal formation than males. Although the wire injury procedure can be performed on either side of the femoral arteries, we prefer the left side because of the anatomical accessibility for right-handed operators.
Among all the mouse models of post-angioplasty restenosis, arterial wire injury mostly closely mimics angioplasty by endothelial denudation and vessel wall overstreching.2,5,6 There are two major target arteries for wire injury: femoral arteries and carotid arteries. From a technical point of view, wire insertion is easier for carotid arteries because they are wider than femoral arteries. However, neointima formation tends to be less robust in carotid arteries than that in femoral arteries, likely due to less effective denudation. Some authors use a handmade device to create a more aggressive endothelial denudation in carotid arteries.7 On the other hand, the advantage of the femoral artery wire injury model is that it can be performed with homogeneously manufactured, commercially available wires to induce neointimal hyperplasia in a reproducible manner.
To make a successful wire injury in mouse femoral arteries, the key is to operate under a dissecting microscope with sufficient magnification and to use fine instruments &#8211; microtweezers and microscissors in particular. Microtweezers with sharp tips are useful to dissect out the tissues surrounding the artery whereas the ones with blunt tips are useful to place a suture loop around the artery and to dilate the wire entrance hole&#160;made by a sharp arteriotomy with microscissors.
An anatomical dissection of the artery is another key. Around the arterial adventitia there exists a thin plane where the dissection can be carried out easily and smoothly. Find the plane first, then proceed with the dissection. Also, try to dissect out the adjacent vein only at the site of suture loops to avoid vein damage, which is the major cause of bleeding in this procedure. Although in the original method the artery and vein are looped together,4 we find that looping artery only gives better control for both hemostasis and wire insertion.
Wire insertion is the most difficult part of this procedure. A 0.015-inch wire is roughly three times thicker than the DFA, the access artery. The key techniques here are: dilating the DFA itself before arteriotomy; making a small entrance hole which is gradually dilated with microtweezer tips; and avoiding forceful wire insertion &#8211; those techniques are detailed in the protocol section. While in the original method lidocaine is applied to dilate the artery before wire insertion4, we find it unnecessary.
Neointima formation after femoral artery wire injury progresses with time, peaks at 3&#8211;4 weeks after injury, and remains stable at 8 weeks.4,8 The authors usually wait for 4 weeks based on our preliminary data that the amount of neointima at 3 weeks is generally less than the amount at 4 weeks after wire injury (data not shown).
Interestingly, the responses to arterial injury differ between various mouse strains. According to several reports investigating differences between mouse strains after femoral or carotid artery injury, FVB/N mice display the highest amount of neointima formation, followed by 129/SvJ, C3H/HeJ, BALB/C, and C57BL/6, respectively.4,9,10 These characteristics should be considered when composing an experimental plan. The femoral injury model can also be utilized for investigation of inflammatory changes after vascular injury. Inflammatory cell infiltration was reported to increase significantly three days after injury.11
In conclusion, the wire injury model using mouse femoral arteries is particularly suitable to investigate post-angioplasty restenosis, and the process can be performed safely and easily using the method presented here. The whole procedure can be finished within 20 min once the techniques are fully familiarized.

In the era of expanded application of endovascular treatments for various cardiovascular diseases, restenosis after angioplasty is one of the major problems for patients undergoing such treatments. Damage to the vascular endothelium at the time of angioplasty, in concert with the atherosclerotic background, induces excessive smooth muscle cell proliferation in the medial layer, resulting in neointimal hyperplasia.1,2 A viable animal model that recapitulates post-angioplasty neointimal hyperplasia is, thus, important not only for the investigation of disease mechanisms but also for the development of effective therapeutics to treat this pathology.
Mice represent an excellent model animal to recapitulate neointimal hyperplasia for the following reasons: the genetic backgrounds of experimental mice are well established; a wide variety of genetically-modified strains are available;2 obtaining littermates of the same background is easy; and the cost of the animals is relatively low. Arterial ligation model and wire injury model are the two most common mouse models of mechanically-induced neointimal hyperplasia. The arterial ligation model is easy to create, but physiologically dissimilar to the actual angioplasty procedure. The wire injury model closely mimics actual angioplasty procedures but is technically difficult due to the small size of mouse arteries.2,3 Sata et al. first described a wire injury method for mouse femoral arteries based on the anatomical structure of the vasculature and the use of proper-sized flexible wire. Utilizing this technique, they succeeded in reproducibly inducing neointimal hyperplasia in various strains of mice.4
Although femoral wire injury is a well-established model, some of the technical aspects of the technique are highly challenging compared to other models such as ligation.5 The purpose of this paper is to describe our mouse wire injury model procedures in detail, which is a modified version of Sata&#8217;s original method. We have made two main modifications: 1) Looping only the arteries, and 2) No lidocaine use.

<table><tbody><tr><td>Fixed Core Wire Guide</td><td>Cook</td><td>G02426</td><td>Diameter 0.015 inch, Straight</td></tr><tr><td>Dilation Forceps</td><td>Roboz</td><td>RS-4927</td><td>Curved, blunt tips</td></tr><tr><td>Dumont Tweesers #5</td><td>World Precision Instruments</td><td>14095</td><td>Straight, sharp tips</td></tr><tr><td>Dumont Vessel Cannulation Forceps</td><td>World Precision Instruments</td><td>503373</td><td></td></tr><tr><td>McPherson-Vannas Scissors</td><td>World Precision Instruments</td><td>501234</td><td></td></tr><tr><td>Mosquito Forceps</td><td>World Precision Instruments</td><td>501291</td><td></td></tr><tr><td>Ethilon Nylon Suture 9-0</td><td>Ethicon</td><td>7717G</td><td>9-0, Black Nylon Monofilament</td></tr><tr><td>Micro AROSuture, Sterile 11-0, 70 micrometers, MET Point</td><td>AROSurgical</td><td>VT4A00N07</td><td>11-0, Black Nylon Monofilament</td></tr><tr><td>3M Precise Multi-Shot DS Disposable Skin Stapler</td><td>3M</td><td>DS-25</td><td></td></tr></tbody></table>

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.

Four weeks following surgery, harvest the wire-injured femoral artery and make a paraffin block specimen to analyze the neointima formation. Induce general anesthesia as described above. Open the chest immediately and insert a 20-gauge butterfly needle to the left ventricle. Connect the line to an infusion bottle. Perform perfusion fixation via drip infusion of PBS followed by 4 % paraformaldehyde (usually 10&#8211;20 ml, each). The infusion bottle is placed approximately 140 cm higher than the heart level so that the animal is perfused at physiological pressure.Wait for 20 min then take out the wire-injured CFA and fix it in 4 % paraformaldehyde overnight at 4 &#176;C. Make a paraffin block of the specimen using standard histological techniques and prepare cross sections of 5-&#956;m thickness for histological staining.
Hematoxylin-eosin staining will allow clear visualization of neointima formation inside the wire-injured femoral artery (Figure 3A, wire-injured femoral artery; 3B, uninjured femoral artery).Use a proper software to measure the lumen area (LA), the internal elastic lamina (IEL) area, and the external elastic lamina (EEL) area from cross section (Figure 3C). Calculate the intima area by subtracting lumen area from IEL area; calculate the media area by subtracting IEL area from EEL area. Intima area/media area ratio is used to compare the neointima formations between different experimental groups.
<img alt="Figure 1" src="/files/ftp_upload/52561/52561fig1highres.jpg" /> 
Figure 1: Schematic Illustrations of Skin Incision and Vasculature Anatomy. (A) An appropriate skin incision is about 1.5 cm in length, along the midline of medial upper thigh. (B) Find the superficial femoral artery first, the common femoral artery next, and finally, the deep femoral artery. Dissect each artery carefully without damaging the adjacent vein.
<img alt="Figure 2" src="/files/ftp_upload/52561/52561fig2highres.jpg" /> 
Figure 2: Wire Injury Procedure. (A) The common femoral artery (arrow), the superficial femoral artery (dotted arrow), and the deep femoral artery (arrow head) are exposed and looped with sutures. (B) Note the size difference between the arteries and the 0.015 inch wire. (C) The wire is inserted via the deep femoral artery. Keep the wire inside for 1 min to have the arterial wall overstretched. (D) Blood flow is resumed. The common femoral artery is well-expanded after wire injury (arrow). Bar indicates 1 mm.
<img alt="Figure 3" src="/files/ftp_upload/52561/52561fig3highres.jpg" />
Figure 3: Hematoxylin-eosin staining of wire-injured and uninjured femoral arteries. (A) A cross-section of a wire-injured femoral artery reveals a thick, highly cellular neointima plaque. (B) A cross-section of an uninjured femoral artery demonstrates intact arterial wall structures without any neointima formation. (C) The green line delineates lumen area, the yellow line delineates internal elastic lamina, and the blue line delineates external elastic lamina. Bar indicates 100 &#956;m.

Watch this Scientific Journal Video about A Murine Model of Arterial Restenosis: Technical Aspects of Femoral Wire Injury at JoVE.com

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

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 ...

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20.1K Views.  University of Wisconsin School of Medicine and Public Health. 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 neointima for studies of restenosis.

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

Murine Model of Hindlimb Ischemia 

In the United States, peripheral arterial disease (PAD) affects about 10 million individuals, and is also prevalent worldwide. Medical therapies for symptomatic relief are limited. Surgical or endovascular interventions are useful for some individuals, but long-term results are often disappointing. As a result, there is a need for developing new therapies to treat PAD. The murine hindlimb ischemia preparation is a model of PAD, and is useful for testing new therapies. When compared to other models of tissue ischemia such as coronary or cerebral artery ligation, femoral artery ligation provides for a simpler model of ischemic tissue. Other advantages of this model are the ease of access to the femoral artery and low mortality rate. 
In this video, we demonstrate the methodology for the murine model of unilateral hindimb ischemia. The specific materials and procedures for creating and evaluating the model will be described, including the assessment of limb perfusion by laser Doppler imaging. This protocol can also be utilized for the transplantation and non-invasive tracking of cells, which is demonstrated by Huang et al.1.

Murine Model of Hindlimb Ischemia

The surgical procedure for induction of unilateral hindlimb ischemia is demonstrated, with confirmation of ischemia by laser Doppler perfusion imaging.

In the United States, peripheral arterial disease (PAD) affects about 10 million individuals, and is also prevalent worldwide.  Medical therapies for ...

Biology

Murine Spinotrapezius Model to Assess the Impact of Arteriolar Ligation on Microvascular Function and Remodeling

The murine spinotrapezius is a thin, superficial skeletal support muscle that extends from T3 to L4, and is easily accessible via dorsal skin incision. Its unique anatomy makes the spinotrapezius useful for investigation of ischemic injury and subsequent microvascular remodeling. Here, we demonstrate an arteriolar ligation model in the murine spinotrapezius muscle that was developed by our research team and previously published1-3. For certain vulnerable mouse strains, such as the Balb/c mouse, this ligation surgery reliably creates skeletal muscle ischemia and serves as a platform for investigating therapies that stimulate revascularization. Methods of assessment are also demonstrated, including the use of intravital and confocal microscopy. The spinotrapezius is well suited to such imaging studies due to its accessibility (superficial dorsal anatomy) and relative thinness (60-200 &mu;m). The spinotrapezius muscle can be mounted en face, facilitating imaging of whole-muscle microvascular networks without histological sectioning. We describe the use of intravital microscopy to acquire metrics following a functional vasodilation procedure; specifically, the increase in arterilar diameter as a result of muscle contraction. We also demonstrate the procedures for harvesting and fixing the tissues, a necessary precursor to immunostaining studies and the use of confocal microscopy.

We demonstrate a novel arterial ligation model in murine spinotrapezius muscle, including a step-by-step procedure and description of required instrumentation. We describe the surgery and relevant outcome measurements relating to vascular network remodeling and functional vasodilation using intravital and confocal microscopy.

The murine spinotrapezius is a thin, superficial skeletal support muscle that extends from T3 to L4, and is easily accessible via dorsal skin incision. ...

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.

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 ...

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

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

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

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

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. ...

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

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. Moreover, by rupture of the defective vascular wall, atherosclerosis induces occlusive thrombus formation, which represents the main cause of myocardial infarction or stroke and the most frequent cause of death. Despite the advances in the cardiovascular field, many questions remain unanswered, and additional basic research is essential to improve our understanding of the molecular mechanisms during atherosclerosis and its effects. Due to limited clinical studies, there is a need for representative animal models recreating atherosclerotic conditions such as neointima formation after stent implantation, balloon angioplasty, or endarterectomy. Since the mouse presents many advantages and is the most frequently used model for studying molecular processes, the current study proposes an invasive procedure of endothelial denudation, also known as the wire-injury model, which is representative of the human condition of neointima formation in arteries after revascularization procedures.

This study describes an invasive procedure for the induction of accelerated atherosclerosis in mice. In comparison to other methods using electric- or cryo-induced injury, mechanical-induced injury mimics the human condition of restenosis after revascularization therapies and is ideal for the study of the molecular mechanisms involved.

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. ...

Reproducible Arterial Denudation Injury by Infrarenal Abdominal Aortic Clamping in a Murine Model

Percutaneous vascular interventions uniformly result in arterial denudation injuries that subsequently lead to thrombosis and restenosis. These complications can be attributed to impairments in re-endothelialization within the wound margins. Yet, the cellular and molecular mechanisms of re-endothelialization remain to be defined. While several animal models to study re-endothelialization after arterial denudation are available, few are performed in the mouse because of surgical limitations. This undermines the opportunity to exploit transgenic mouse lines and investigate the contribution of specific genes to the process of re-endothelialization. Here, we present a step-by-step protocol for creating a highly reproducible murine model of arterial denudation injury in the infrarenal abdominal aorta using external vascular clamping. Immunocytochemical staining of injured aortas for fibrinogen and &#946;-catenin demonstrate the exposure of a pro-thrombotic surface and the border of intact endothelium, respectively. The method presented here has the advantages of speed, excellent overall survival rate, and relative technical ease, creating a uniquely practical tool for imposing arterial denudation injury in transgenic mouse models. Using this method, investigators may elucidate the mechanisms of re-endothelialization under normal or pathological conditions.

Understanding the cellular and molecular mechanisms of re-endothelialization following arterial denudation injury is of paramount importance in preventing thrombosis and restenosis of arteries. Here we describe a protocol for reproducible arterial denudation injury of the infrarenal abdominal aorta. The procedure was developed to investigate the underlying mechanisms that regulate endothelial regeneration using mouse models.

Percutaneous vascular interventions uniformly result in arterial denudation injuries that subsequently lead to thrombosis and restenosis. These ...

A Rat Carotid Artery Pressure-Controlled Segmental Balloon Injury with Periadventitial Therapeutic Application

Cardiovascular disease remains the leading cause of death and disability worldwide, in part due to atherosclerosis. Atherosclerotic plaque narrows the luminal surface area in arteries thereby reducing adequate blood flow to organs and distal tissues. Clinically, revascularization procedures such as balloon angioplasty with or without stent placement aim to restore blood flow. Although these procedures reestablish blood flow by reducing plaque burden, they damage the vessel wall, which initiates the arterial healing response. The prolonged healing response causes arterial restenosis, or re-narrowing, ultimately limiting the long-term success of these revascularization procedures. Therefore, preclinical animal models are integral for analyzing the pathophysiological mechanisms driving restenosis, and provide the opportunity to test novel therapeutic strategies. Murine models are cheaper and easier to operate on than large animal models. Balloon or wire injury are the two commonly accepted injury modalities used in murine models. Balloon injury models in particular mimic the clinical angioplasty procedure and cause adequate damage to the artery for the development of restenosis. Herein we describe the surgical details for performing and histologically analyzing the modified, pressure-controlled rat carotid artery balloon injury model. Additionally, this protocol highlights how local periadventitial application of therapeutics can be used to inhibit neointimal hyperplasia. Lastly, we present light sheet fluorescence microscopy as a novel approach for imaging and visualizing the arterial injury in three-dimensions.

The rat carotid artery balloon injury mimics the clinical angioplasty procedure performed to restore blood flow in atherosclerotic vessels. This model induces the arterial injury response by distending the arterial wall, and denuding the intimal layer of endothelial cells, ultimately causing remodeling and an intimal hyperplastic response.

Cardiovascular disease remains the leading cause of death and disability worldwide, in part due to atherosclerosis. Atherosclerotic plaque narrows the ...

Multiphoton Intravital Imaging for Monitoring Leukocyte Recruitment during Arteriogenesis in a Murine Hindlimb Model

Arteriogenesis strongly depends on leukocyte and platelet recruitment to the perivascular space of growing collateral vessels. The standard approach for analyzing collateral arteries and leukocytes in arteriogenesis is ex vivo (immuno-) histological methodology. However, this technique does not allow the measurement of dynamic processes such as blood flow, shear stress, cell-cell interactions, and particle velocity. This paper presents a protocol to monitor in vivo processes in growing collateral arteries during arteriogenesis utilizing intravital imaging. The method described here is a reliable tool for dynamics measurement and offers a high-contrast analysis with minimal photo-cytotoxicity, provided by multiphoton excitation microscopy. Prior to analyzing growing collateral arteries, arteriogenesis was induced in the adductor muscle of mice by unilateral ligation of the femoral artery.
After the ligation, the preexisting collateral arteries started to grow due to increased shear stress. Twenty-four hours after surgery, the skin and subcutaneous fat above the collateral arteries were removed, constructing a pocket for further analyses. To visualize blood flow and immune cells during in vivo imaging, CD41-fluorescein isothiocyanate (FITC) (platelets) and CD45-phycoerythrin (PE) (leukocytes) antibodies were injected intravenously (i.v.) via a catheter placed in the tail vein of a mouse. This article introduces intravital multiphoton imaging as an alternative or in vivo complementation to the commonly used static ex vivo (immuno-) histological analyses to study processes relevant for arteriogenesis. In summary, this paper describes a novel and dynamic in vivo method to investigate immune cell trafficking, blood flow, and shear stress in a hindlimb model of arteriogenesis, which enhances evaluation possibilities notably.

The recruitment of leukocytes and platelets constitutes an essential component necessary for the effective growth of collateral arteries during arteriogenesis. Multiphoton microscopy is an efficient tool for tracking cell dynamics with high spatio-temporal resolution in vivo and less photo-toxicity to study leukocyte recruitment and extravasation during arteriogenesis.

Arteriogenesis strongly depends on leukocyte and platelet recruitment to the perivascular space of growing collateral vessels. The standard approach for ...

Murine Aortic Crush Injury: An Efficient In Vivo Model of Smooth Muscle Cell Proliferation and Endothelial Function

Arterial reconstruction, whether angioplasty or bypass surgery, involves iatrogenic trauma causing endothelial disruption and vascular smooth muscle cell (VSMC) proliferation. Common murine models study small vessels such as the carotid and femoral arteries. Herein we describe an in vivo system in which both VSMC proliferation and endothelial barrier function can be simultaneously assessed in a large vessel. We studied the infrarenal aortic response to injury in C57BL/6 mice. The aorta was injured from the left renal vein to the aortic bifurcation by 30 transmural crushes of 5-seconds duration with a cotton-tipped applicator. Morphological changes were assessed with conventional histology. Aorta wall thickness was measured from the luminal surface to the adventitia. EdU integration and counter staining with DAPI and alpha-actin was used to demonstrate VSMC proliferation. Activation of ERK1/2, a known moderator of intimal hyperplasia formation, was determined by Western Blot analysis. The effect of inflammation was determined by immunohistochemistry for B-cells, T-cells, and macrophages. En face sections of endothelium were visualized with scanning electron microscopy (SEM). Endothelial barrier function was determined with Evans Blue staining. Transmural injury resulted in aortic wall thickening. This injury induced VSMC proliferation, most prominently at 3 days after injury, and early activation of ERK1/2 and decreased p27kip1 expression. Injury did not result in increased B-cells, T-cells, or macrophages infiltration in the vessel wall. Injury caused partial endothelial cell denudation and loss of cell-cell contact. Injury resulted in a significant loss of endothelial barrier function, which returned to baseline after seven days. The murine transmural blunt aortic injury model provides an efficient system to simultaneously study both VSMC proliferation and endothelial barrier function in a large vessel.

Murine Aortic Crush Injury: An Efficient In Vivo Model of Smooth Muscle Cell Proliferation and Endothelial Function

Restenosis following cardiovascular procedures (bypass surgery, angioplasty, or stenting) is a significant problem reducing the durability of these procedures. An ideal therapy would inhibit smooth muscle cell (VSMC) proliferation while promoting regeneration of the endothelium. We describe a model for simultaneous assessment of VSMC proliferation and endothelial function in vivo.

Arterial reconstruction, whether angioplasty or bypass surgery, involves iatrogenic trauma causing endothelial disruption and vascular smooth muscle cell ...

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

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Chapters in this video

Murine Model of Hindlimb Ischemia

Murine Spinotrapezius Model to Assess the Impact of Arteriolar Ligation on Microvascular Function and Remodeling

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

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

Murine Model of Femoral Artery Wire Injury with Implantation of a Perivascular Drug Delivery Patch

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

Reproducible Arterial Denudation Injury by Infrarenal Abdominal Aortic Clamping in a Murine Model

A Rat Carotid Artery Pressure-Controlled Segmental Balloon Injury with Periadventitial Therapeutic Application

Multiphoton Intravital Imaging for Monitoring Leukocyte Recruitment during Arteriogenesis in a Murine Hindlimb Model

Murine Aortic Crush Injury: An Efficient In Vivo Model of Smooth Muscle Cell Proliferation and Endothelial Function