Name: A Murine Model of Arterial Restenosis: Technical Aspects of Femoral Wire Injury (Video) | JoVE Journal
Uploaded: 2015-03-10T15:00:00.000+00:00
Duration: 6 min 42 s
Description: Learn the femoral artery wire injury technique in mice to induce intimal hyperplasia for restenosis research. This protocol demonstrates precise surgical steps and histological validation methods.

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5 minutes

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/pubmed?term=((Vascular+proliferation+and+atherosclerosis%3A+new+perspectives+and+therapeutic+strategies%5BTitle%5D)+AND+%22Nat+Med%22%5BJournal%5D)">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/pubmed?term=((Intimal+hyperplasia+in+murine+models%5BTitle%5D)+AND+%22Curr+Drug+Targets%22%5BJournal%5D)">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/pubmed?term=((The+mechanisms+of+coronary+restenosis%3A+insights+from+experimental+models%5BTitle%5D)+AND+%22Int+J+Exp+Pathol%22%5BJournal%5D)">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/pubmed?term=((A+mouse+model+of+vascular+injury+that+induces+rapid+onset+of+medial+cell+apoptosis+followed+by+reproducible+neointimal+hyperplasia%5BTitle%5D)+AND+%22J+Mol+Cell+Cardiol%22%5BJournal%5D)">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/pubmed?term=((Mouse+models+of+arteriosclerosis%3A+from+arterial+injuries+to+vascular+grafts%5BTitle%5D)+AND+%22Am+J+Pathol%22%5BJournal%5D)">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/pubmed?term=((STAT4+deficiency+protects+against+neointima+formation+following+arterial+injury+in+mice%5BTitle%5D)+AND+%22J+Mol+Cell+Cardiol%22%5BJournal%5D)">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/pubmed?term=((Apolipoprotein+E+inhibits+neointimal+hyperplasia+after+arterial+injury+in+mice%5BTitle%5D)+AND+%22Am+J+Pathol%22%5BJournal%5D)">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/pubmed?term=((Effect+of+metabolic+syndrome+on+the+response+to+arterial+injury%5BTitle%5D)+AND+%22J+Surg+Res%22%5BJournal%5D)">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/pubmed?term=((Distinction+in+genetic+determinants+for+injury-induced+neointimal+hyperplasia+and+diet-induced+atherosclerosis+in+inbred+mice%5BTitle%5D)+AND+%22Arterioscler+Thromb+Vasc+Biol%22%5BJournal%5D)">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/pubmed?term=((Mouse+strain+differential+neointimal+response+in+vein+grafts+and+wire-injured+arteries%5BTitle%5D)+AND+%22Circ+J%22%5BJournal%5D)">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/pubmed?term=((Deficiency+of+CuZn+superoxide+dismutase+promotes+inflammation+and+alters+medial+structure+following+vascular+injury%5BTitle%5D)+AND+%22J+Atheroscler+Thromb%22%5BJournal%5D)">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 t...

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

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 10–20 ml, each). The infusion bottle is placed approximately 140 cm higher than the heart level so that the...

The mouse femoral artery wire injury model of restenosis is technically challenging. In this protocol we show the key technical details essential for successfully performing wire injury to induce consistent&#160;neointima for studies of restenosis.

Cardiovascular disease caused by atherosclerosis is the leading cause of death in the developed world. Narrowing of the vessel lumen, due to ...

The goal of this procedure is to create prominent intimal hyperplasia induced by wire injury in the femoral artery in mice. This is accomplished by first making a longitudinal skin incision on the left thigh. Next, the femoral artery is found and looped at three important anatomical regions, the superficial femoral artery or SFA common femoral artery, or CFA and deep femoral artery, or DFA.Then a small hole is made on the DFA wall and a metallic wire is inserted and advanced into the CFA where it is left for one minute. Finally, the wire is retrieved and the DFA is ligated at two points. Ultimately, h and e staining of cross-sections shows thick intimal hyperplasia in the femoral artery.Generally, individuals new to this method will struggle because of two reasons. First, they have to find the right access artery from deep inside the muscle. Second, they have to insert a wire, which is roughly three times thicker than the access artery After anesthetizing a male C 57 plaque six mouse.According to the text protocol, confirming proper anesthesia and applying eye ointment place the mouse in the supine position, the left limb slightly abducted and the knee joints slightly flexed. Once the hair has been shaved and the region has been disinfected with fine tweezers, open the thin fascia overlying the superficial femoral artery, and use nine zero nylon suture. To loop the SFA use mosquito forceps to hold the suture and gently pull it slightly downward to make further dissection easier.Continue to dissect the artery to expose the common femoral artery or CFA. Then use nine zero nylon suture. To loop the CFA while holding both suture loops.Pull the CFA suture loop upward and the SFA suture loop downward to uncover the bifurcation of the SFA and the deep femoral artery or DFA, which branches towards the thigh muscle. Next, using extra caution loop the DFA taking care to avoid the adjacent vein that is tangled beneath the artery. In case of bleeding from the vein, use GREs to apply a 30 to 62nd compression to produce hemostasis.Once the DFA is looped, make sure all three suture loops are at their correct positions. Then proceed to dissect the DFA distally and ligate the artery at a point as far away as possible from the bifurcation. Cut, extra suture, but leave a certain length for using mosquito forceps to hold the two ends to stabilize the DFA, which will make the wire insertion easier to insert the wire via the DFA, begin by straining the C-F-A-S-F-A and DFA sutures.To establish a temporary hemostat, loosen the CFA suture loop and use dull tipped micro tweezers to gently clamp or squeeze the DFA several times. This will expand the arterial diameter remarkably to ease wire insertion. Next with sharp tipped micro tweezers, make a small V-shaped nip in the DFA and pinch the flap.Use PBS to moisten the wire thoroughly and without using force. Insert it through the adequately dilated hole on the DFA gently advance the wire toward the iliac artery. While loosening the suture loop around the CFA five to eight millimeters is enough to cover the length of the CFA in most cases.After inserting the wire to the optimal length, leave it for one minute to overstretch the arterial wall, use PBS to moisten the wire and arteries to prevent drying while waiting. Place another nine zero suture around the DFA proximal to the wire insertion point to be used later. To close the hole of the DFA to retrieve the wire and resume blood flow.Use vessel cannulation forceps to hold the wire and gently pull it back, rotating it if necessary to overcome any resistance due to sticking to the arterial wall. After retrieving the wire, use nine zero suture to ligate the DFA as a point closer to the bifurcation, ensuring that blood outflow to the SFA will not be jeopardized. Loosen the SFA and CFA suture loops to resume normal blood flow.The CFA will be well expanded by the wire insertion, close the skin, carry out postoperative care, and later harvest the wire injured femoral artery. According to the text protocol, after harvesting the femoral artery, the tissue was fixed in paraldehyde and embedded in param shown. Here are h and e stain slices of the embedded tissue.Neoma formation can be clearly seen inside the wire injured femoral artery. The lumen area internal elastic lamina, or IEL area, and the external elastic lamina or EEL area from the cross section are indicated in this panel. These values can be used to calculate the intimate area by subtracting the lumen area from the IEL area and the media area.By subtracting the IEL area from the EEL area, the intimate area media area ratio is used to compare the neointima formations between different experimental groups. Once mastered, this technique can be done in 20 minutes if it is performed properly.

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Learn the femoral artery wire injury technique in mice to induce intimal hyperplasia for restenosis research. This protocol demonstrates precise surgical steps and histological validation methods.

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

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