The authors would like to acknowledge support through the American Heart Association (10SDG2630139), the Welch Foundation and through the NIH Director&#8217;s New Innovator Grant (1DP2 OD008716-01). The authors would like to thank the services provided by the ICMB (Institute of Cellular and Molecular Biology) core facility and TherapeUTex at University of Texas at Austin.

NOTE: All methods shown in this protocol have been approved by the Institutional Animal Care and Use Committee.
1. Preparation of Surgical Table
<ol>
	<li>Set up the heating pad with recirculating warm water on the surgical table, under the dissection microscope. Place the stainless steel base plate onto the heating pad. Place a sterile absorbent pad on the base plate and heating pad.</li>
	<li>Arrange sterile surgical tools on an adjacent sterile absorbent pad. Gather two pairs of angled fine-tipped forceps, two pairs of angled forceps, three hemostatic forceps, two retractors&#160;wires, small surgical scissors, micro-scissors, one angioplasty wire (0.15 inch diameter)&#160;and three 6-inch segments of 6.0 silk suture.</li>
	<li>Bend the round end of the angioplasty wire to match the curvature of the femoral artery. This will make it easier to advance the wire during the surgery.</li>
</ol>
2. Preparation of Mouse for Surgery
<ol>
	<li>Anesthetize a mouse by continuous inhalation of 2.5% isoflurane. Be sure to monitor the animal&#8217;s status throughout the procedure. Perform a pinch test of the mouse&#8217;s foot to confirm that it is fully anesthetized. Ensure the animal does not move when the pinch test is administered.</li>
	<li>Apply a lubricating ointment to the animal&#8217;s eyes to prevent desiccation. Secure the animal in the supine position to the absorbent pad using surgical tape. Continue administering 2.5% isoflurane via nose cone.</li>
	<li>Using a depilatory cream, remove fur from the leg and abdomen to the mid-line. Rinse the skin thoroughly with water. Depilation can be performed one day in advance of surgery. Note that excessive treatment with the cream may lead to skin irritation.</li>
	<li>Immediately before surgery, apply povidone-iodine to the depilated area with a cotton-tipped applicator to sterilize the skin. Rinse the depilated skin with 70% ethanol and dry with a sterile cotton-tipped applicator. &#160;Repeat 3 times.&#160;</li>
	<li>Administer a pre-operative dose of 5 mg/kg of carprofen via subcutaneous injection.</li>
</ol>
3. Isolation of the Femoral Artery
<ol>
	<li>Using small surgical scissors, make a curved incision in the skin over the femoral artery. Bluntly dissect and secure surrounding tissues using retractors magnetic retractor fixators to locate the femoral artery. Moisten the tissues periodically using saline for irrigation Apply saline using a sterile cotton-tipped applicator.</li>
	<li>Isolate the femoral artery using forceps. Gently separate the nerve from the vascular bundle using fine-tipped forceps. Avoid puncturing the vein, and do not damage the nerve. Push the nerve away from the bundle to avoid stimulating it.</li>
	<li>Gently separate the femoral vein from the femoral artery, locating the femoral bifurcation. The region of the bifurcation is especially difficult to dissect.</li>
	<li>Posterior to the bifurcation, loop a 6.0 silk suture under the femoral artery and secure with a hemostat. This proximal suture will be used to restrict blood flow in the artery. 
	NOTE: There is a slight variation in the ties when performing the ligation method versus the cauterization method (see Figure 1 and Figure 2).</li>
	<li>Distal to the bifurcation, loop 6.0 silk suture under the femoral artery and secure with a hemostat. This distal suture aids in the positioning of the artery.</li>
	<li>Loop two sutures under the muscular branch of the femoral artery, pre-tie them and secure with a hemostat. Remember to moisten the tissues with saline. If performing the cautery method of wire injury, only one looped suture is necessary on the muscular branch.</li>
</ol>
4. Performance of Femoral Artery Wire Injury
<ol>
	<li>Restrict blood flow into the femoral artery by pulling the proximal suture. Slightly pull the distal hemostat and hemostat securing the branch to expose the site for the arteriotomy. Ligate the muscular branch by tying the suture around it. 
	NOTE: Lifting the artery upward will more effectively restrict blood flow than pulling horizontally alone.</li>
	<li>Sever the small branch with the cautery between the two sutures. Using micro-scissors, perform an arteriotomy in the side branch of the bifurcation. Use forceps balanced on a roll of surgical tape to stabilize the micro-scissors.</li>
	<li>Confirm the presence of the arteriotomy using fine-tipped forceps. Gently lift the opening of the arteriotomy with the forceps. Introduce the rounded end of the wire into the arteriotomy using forceps. To ease advancement of the wire, add one or two drops of lidocaine to the region using a syringe.</li>
	<li>When the wire reaches the proximal suture, release the suture and adjust it so that it cannot impede the advancement of the wire. Insert the wire until it cannot advance further. The tip of the wire should stop in the region of the inguinal ligament.</li>
	<li>Allow the wire to remain in the femoral artery for one minute. After one minute, retract and advance the wire in a sawing motion ten times to injure and denude the endothelium of the femoral artery. For an injury of lesser severity, decrease the number of times the wire is pulled in and out of the artery or leave the wire in the artery for 1 min only.</li>
	<li>Retract the wire slowly. When the round end of the wire has passed the proximal suture, restrict flow into the artery by pulling the proximal suture. Retract the wire completely.</li>
</ol>
5. Ligating the Muscular Branch
<ol>
	<li>Tighten the remaining suture on the muscular branch. This will prevent bleeding from the arteriotomy. Return flow to the muscular branch, and confirm that blood is not leaking from it. Trim the ends from the sutures on the muscular branch.</li>
</ol>
6. Alternative Method: Localized Cauterization of the Arteriotomy
NOTE: An alternative approach can be taken to avoid ligation of the muscular branch and allow vascular access through the larger main femoral artery.
<ol>
	<li>Starting from 4.1, restrict flow to the femoral artery by pulling on the proximal suture. Pull the distal suture and the suture securing the muscular branch slightly to expose the site for the arteriotomy (Figure 1 and Figure 2).</li>
	<li>Using micro-scissors, perform an arteriotomy at the branch point of the femoral artery and the muscular branch. If the incision is made in the side of the artery, it may be easier to cauterize. Use forceps balanced on a roll of surgical tape to stabilize the micro-scissors.</li>
	<li>To test the success of cauterization, restore flow to the femoral artery by loosening the proximal suture. If bleeding occurs from the arteriotomy, repeat the cautery. If the cautery is successful, blood flow will be restored distal to the arteriotomy.</li>
	<li>Introduce the wire and perform the wire injury as described in section 4.</li>
	<li>Continue restricting blood flow into the femoral artery. Heat a fine-tipped cautery at least 6 inches away from the mouse. As the cautery tip cools, apply it lightly side of the arteriotomy to close the incision.</li>
	<li>To test the success of cauterization, restore flow to the femoral artery by loosening the proximal suture. If bleeding occurs from the arteriotomy, repeat the cautery. If the cautery is successful, blood flow will be restored distal to the arteriotomy. Remove the temporary proximal and distal ties.</li>
</ol>
7. Implantation of Perivascular Drug Delivery Patch
<ol>
	<li>Create a perivascular drug patch can be created as described in previous studies11 or by using similar methods. Blunt the corners of the barrier region of the drug delivery patch using sterile scissors</li>
	<li>Place the drug delivery patch onto the injured femoral artery with the drug-releasing side facing the artery. As necessary, use forceps to improve the position of the patch.</li>
</ol>
8. Wound Closure and Recovery
<ol>
	<li>Close the wound with a simple interrupted suture consisting of square knots. A discontinuous suture prolongs wound closure in case the animal tries to remove the suture.</li>
	<li>Turn off the anesthesia and remove the animal from the set up. Allow the animal to recover on the warming pad.</li>
	<li>Continue to monitor the mouse&#8217;s recovery. Check the incision site each day to ensure that it remains closed. For treatment of post-surgical pain, administer a subcutaneous injection of 5 mg/kg of carprofen every 12 hr after surgery and then every 12 hr for 2 days.&#160;If pain continues beyond the initial 2 days, consult a veterinarian for directions on further pain medication.</li>
</ol>
9. Harvesting Femoral Arteries for Histology
<ol>
	<li>At 28 days post-surgery, perform carbon dioxide euthanasia on the mouse. 
	NOTE: The appropriate flow rate of carbon dioxide should displace 10-30% of the chamber per minute and will vary depending on the size of the chamber used. Cutting of the diaphragm and cardiac puncture should be performed as a secondary method of euthanasia.</li>
	<li>Secure the mouse in the supine position using surgical tape. Make an incision over the femoral artery, where the initial surgical incision was made.</li>
	<li>For both the injured artery and the uninjured artery from the contralateral leg, bluntly dissect and secure surrounding tissues using retractors and magnetic retractor fixators to locate the femoral artery. Moisten the tissues periodically using saline for irrigation. Apply saline using a sterile cotton-tipped applicator. Take care to not damage the artery.</li>
	<li>After the femoral artery has been isolated from the site of the original arteriotomy to the abdominal aorta, tie a segment of silk suture near the original site of the arteriotomy. This suture will help identify the most distal end of the femoral artery and ease handling of the sample.</li>
	<li>Using the micro-dissection scissors to excise the femoral artery. Make one incision distal to the suture. Make the other incision on the opposite end of the femoral artery, beside the abdominal aorta.</li>
	<li>Transfer the excised artery to a glass petri dish containing saline. Dissect the artery further to remove excess connective tissue or fat. Gently remove blood from the lumen by rinsing with saline.</li>
	<li>Transfer the artery to a vial of 10% buffered formalin. Store the vial at 4 &#176;C with gentle rocking for 48 hr.</li>
	<li>Transfer the fixed artery to 70% ethanol, to store until it is processed for histology.</li>
	<li>Embed the artery in paraffin blocks and section the blocks for staining.</li>
	<li>Perform histochemical and immunochemical staining to assess the extent of injury and intimal hyperplasia. 
	NOTE: For the representative results, we used Hematoxylin and Eosin to visualize the nuclei and overall morphology or a Movat&#8217;s Pentachrome stain to visualize the elastic lamellae and other arterial components.</li>
</ol>

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We have presented a method for performing vascular injury in mice and delivery therapeutic compounds to the injured region through a perivascular cuff. The ligation method for femoral and carotid arteries has been described in conventional methods papers and characterized extensively11-14,19-23 and we present an alternative method for achieving the same vascular injury that is less technically demanding procedure that can often be used in younger mice. One of the chief advantages of the using a mouse wire injury model with a perivascular patch is that it allows the use of genetically modified mice or murine models of disease. The endovascular manipulation of small arteries is technically demanding and this aspect has motivated the creation of models that create injury through a number of extravascular methods including ligation,11,24,25 perivascular cuffing26 and electric shock.27 In our procedures, a contralateral sham surgery can be used as a control by performing the protocol without the wire injury or, alternatively, non-operated control arteries can be used. The critical steps within the protocol are the following: (1) the surgical cut down and dissection of the femoral artery from surrounding structures, (2) the formation of the arteriotomy, (3) performance of the wire injury, (4) closure of the arteriotomy and (5) implantation of the perivascular cuff. Below we will review the major steps and address potential pitfalls in their performance. 
For the surgical cut down, the major challenge is the separation of the femoral artery from the femoral vein. Care should be taken at this stage, as it is easy to cause bleeding during the separation and the vein tears easily in comparison to the artery. Using forceps to blunt dissect and remove adventitia surround the artery and vein can help this process (see techniques shown in the video). Also, the artery may have branches underneath that can be torn if an overly aggressive technique is used. 
For the formation of the arteriotomy and wire injury, it is critical to use high quality microsurgical scissors. It is often helpful to cut at a 45-degree angle to the axis of the artery and to stabilize the cut with forceps as shown in the video. Too much or too little tension on the artery can make cutting difficult and it is sometimes helpful pull the distal tie before the proximal tie to fill the artery with blood to aid in the performing the arteriotomy. After the cut, opening the hole with the closed point of the scissors or forceps can aid in allowing entry of the wire. The size of the arteriotomy should cut approximately half way through the artery. Again, too much tension on the artery will make insertion of the wire difficult and may tear the vessel. A low to moderate level of tension should be used with the ties. When inserting the wire a slight twisting motion can be helpful but too much twisting will cause the artery to seize, preventing advancement of the wire.
A key step in performing our alternative method is the controlled local cautery that seals the vessel without blocking flow. The cautery should be heated away from the tissue and then touched to the side of the artery. Heating with the cautery while touching the vessel will burn surrounding tissues including the vein and femoral artery branches. One potential limitation of this technique is that if the arteries are not consistently cauterized there may be variations in the flow following the surgery or in the amount of inflammation caused by the burn. Analysis of the artery away from this region and consistent technique can minimize the potential impact of these limitations. We hope this alternative method will make the wire injury surgery more accessible and reduce the need for older mice for vascular injury studies.

Arterial injury and inflammation caused by angioplasty and stent implantation can induce neointimal hyperplasia that contributes to the thickening of the arterial wall, a process known as restenosis.1,2 The formation of restenosis is major mode of failure for interventions such as angioplasty and stenting with bare metal stents.3 Due to recent concerns with the inhibition of vascular healing in arteries treated with drug eluting stents, there is also a need to find compounds that can inhibit restenosis while maintaining vascular healing and re-endothelialization.4-7 In addition, while stents have had success in the coronary vasculature, percutaneous interventions of all types in the peripheral arteries continue to fail at a higher rate due to restenosis.8-10 Mouse models of surgical interventions allow the use of powerful genetic manipulations that can provide mechanistic insight into the mechanisms underlying the failure of clinical therapies and can provide an initial test bed for compounds to inhibit intimal hyperplasia. 
Here, we describe a mouse model of vascular injury that allows the testing of therapeutic compounds to inhibit neointimal hyperplasia and assess whether their effects on re-endothelialization following endothelial denudation. A key challenge in executing vascular injury in mice is the technical skill needed to obtain vascular access and to restore flow to the injured artery following the wire injury. For this reason, simple arterial ligation models have been used to study neointimal hyperplasia in mice that do not require endovascular manipulations but are easier to implement.11 However, this type of surgical model differs substantially from the mechanical and biological aspects of a percutaneous intervention, lacking key aspects including arterial wall stretch, endothelial denudation and luminal blood flow following injury. 
We present two methods for obtaining and closing vascular access for wire injury of the femoral artery in mice. The first technique is the conventional method described by several groups previously and uses vascular access through a side branch of the femoral artery.12-14 This method requires older, larger mice and more surgical skill to implement the endoluminal access through the smaller artery. It also requires the ligation of the muscular branch of the femoral artery following the procedure. The second method we describe uses an arteriotomy in the branch point of the main and side branch and thereby allows for a larger access to the artery for performing wire injury. In this method, the arteriotomy is closed using controlled local cauterization that leaves both branches with blood flow following the procedure. The conventional method is applicable to mice of at least 20 weeks of age while the alternative method can be used in mice of at least 15 weeks of age. In both methods, the wire creates arterial stretch and abrasion leading to injury and endothelial denudation. Following either procedure a perivascular drug delivery patch can be implanted that allows the delivery of compounds to alter the response to injury. The use of the drug delivery patch allows mice to be used as a test bed for new compounds to inhibit restenosis through perivascular therapies.15,16

<table><tbody><tr><td>Straight spring wire, 0.15&amp;rdquo; diameter</td><td>Cook</td><td>G02426</td><td></td></tr><tr><td>High Temperature cautery</td><td>Bovie Medical Corp.</td><td>HIT1</td><td></td></tr><tr><td>High-temperature fine tip for cautery</td><td>Bovie Medical Corp.</td><td>H101</td><td></td></tr><tr><td>Micro-scissors</td><td>Fine Science Tools</td><td>15000-13</td><td>For performance of arteriotomy</td></tr><tr><td>Angled fine-tipped forceps</td><td>Fine Science Tools</td><td>11251-35</td><td>For blunt dissection of vascular bundle</td></tr><tr><td>Angled forceps</td><td>Roboz</td><td>RS-5069</td><td>For clearing tissues</td></tr><tr><td>Surgical scissors</td><td>Roboz</td><td>RS-5840</td><td>For cutting skin</td></tr><tr><td>Retractor</td><td>Fine Science Tools</td><td>18200-10</td><td></td></tr><tr><td>Retractor wire</td><td>Fine Science Tools</td><td>18200-05</td><td>Attached to retractor</td></tr><tr><td>Base plate</td><td>Fine Science Tools</td><td>18200-03</td><td>For use with retractor</td></tr><tr><td>Magnetic retractor fixator</td><td>Fine Science Tools</td><td>18200-01</td><td></td></tr><tr><td>Needle Holder</td><td>Roboz</td><td>RS-7822</td><td></td></tr><tr><td>Hemostatic forceps</td><td>Biomedical Research Instruments, Inc.</td><td>34-1000</td><td></td></tr><tr><td>Dissecting microscope</td><td>Meiji Techno</td><td>EMZ-5TR</td><td></td></tr><tr><td>Microscope light source</td><td>Meiji Techno</td><td>FT191</td><td></td></tr><tr><td>Warm water recirculator</td><td>Gaymar</td><td>TP-500</td><td>For maintaining mouse body temperature</td></tr><tr><td>Reusable heating pad</td><td>Gaymar</td><td>TP-R 22G</td><td>For maintaining mouse body temperature</td></tr><tr><td>Lidocaine</td><td>Various</td><td></td><td></td></tr><tr><td>4.0 Vicryl suture with half circle needle</td><td>Ethicon</td><td>J494G</td><td>For post-surgical wound closure</td></tr><tr><td>Sterile cotton-tipped applicators</td><td>Puritan</td><td>25-806-2WC</td><td>For application of depilatory cream and absorbing fluids</td></tr><tr><td>Depilatory cream</td><td>Nair</td><td></td><td></td></tr><tr><td>Isoflurane</td><td>Various</td><td></td><td></td></tr><tr><td>Betadine</td><td>Various</td><td></td><td></td></tr><tr><td>70% ethanol</td><td>Various</td><td></td><td></td></tr><tr><td>6.0 braided silk suture</td><td>Teleflex Medical</td><td>4-S</td><td>For isolation of femoral artery during surgery</td></tr><tr><td>0.9% sodium chloride</td><td>Various</td><td></td><td>For irrigating tissues</td></tr><tr><td>Gel eye lubricant</td><td>Various</td><td></td><td></td></tr><tr><td>Glass Petri dish</td><td>Pyrex</td><td>3160-60</td><td>For femoral artery harvest</td></tr><tr><td>10% buffered formalin</td><td>Various</td><td></td><td>For fixation of femoral artery</td></tr><tr><td>70% ethanol</td><td>Various</td><td></td><td>For fixation of femoral artery</td></tr><tr><td>Bouin&#039;s fluid</td><td>Electron Microscopy Sciences</td><td></td><td>For Movat&#039;s Pentachrome staining</td></tr><tr><td>Alcian blue, 1%</td><td>Electron Microscopy Sciences</td><td>26385-01</td><td>For Movat&#039;s Pentachrome staining</td></tr><tr><td>Alkaline alcohol</td><td>Electron Microscopy Sciences</td><td>26385-02</td><td>For Movat&#039;s Pentachrome staining</td></tr><tr><td>Orcein, 0.2%</td><td>Electron Microscopy Sciences</td><td>26385-03</td><td>For Movat&#039;s Pentachrome staining</td></tr><tr><td>Hematoxylin alcoholic, 5%</td><td>Electron Microscopy Sciences</td><td>26385-04</td><td>For Movat&#039;s Pentachrome staining</td></tr><tr><td>Ferric chloride, 10%</td><td>Electron Microscopy Sciences</td><td>26385-05</td><td>For Movat&#039;s Pentachrome staining</td></tr><tr><td>Lugol&#039;s Iodine</td><td>Electron Microscopy Sciences</td><td>26385-06</td><td>For Movat&#039;s Pentachrome staining</td></tr><tr><td>Woodstain scarlet-acid fuchsin working solution</td><td>Electron Microscopy Sciences</td><td>26385-07</td><td>For Movat&#039;s Pentachrome staining</td></tr><tr><td>Acetic acid, 0.5%</td><td>Electron Microscopy Sciences</td><td>Various</td><td>For Movat&#039;s Pentachrome staining</td></tr><tr><td>Phosphotungstic acid, 5%</td><td>Electron Microscopy Sciences</td><td>26385-09</td><td>For Movat&#039;s Pentachrome staining</td></tr><tr><td>Alcoholic saffron, 6%</td><td>Electron Microscopy Sciences</td><td>26385-10</td><td>For Movat&#039;s Pentachrome staining</td></tr></tbody></table>

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.

Following wire injury, neointimal hyperplasia develops over time and is typically examined after 14 to 28 days. The techniques described in this work lead to robust generation of intimal hyperplasia in mice as shown the histological results in Figure 3. An uninjured femoral artery will demonstrate intact elastic lamellae and a normal thickness and circumference. An injured femoral artery will show intimal hyperplasia, degraded elastic lamellae and demonstrate re-endothelialization at later time points. Re-endothelialization is typically complete at around 21 days but this depends on the background strain of the mice used and can be assessed using immunostaining for endothelial markers on sections or, preferably, measured on en face preparations using the Evan&#8217;s blue dye, scanning electron microscopy or immunostaining with confocal microscopy on en face preparations of the artery.17,18 For quantification of intimal hyperplasia it is best to measure the intimal area or intimal-to-media ratio at three sites along the injured artery to account for potential variations along the length of the injured region of the artery.
<img alt="Figure 1" src="/files/ftp_upload/52403/52403fig1highres.jpg" /> 
Figure 1: Photographs of the critical steps of the two methods for wire injury in the femoral artery. A comparison of the steps for the ligation method (top) to the cautery method (bottom) for performing wire injury. In the ligation method, the two sutures securing the muscular branch are pre-tied so they can be tightened to ligate the muscular branch. The more distal of the two muscular branch sutures is tightened. The arteriotomy and wire insertion are performed in the muscular branch. After the injury is performed, the remaining suture on the muscular branch is tightened and trimmed. In the cautery method, only one suture is looped under the muscular branch. The arteriotomy and wire insertion are performed at the branch point of the muscular branch from the femoral artery. After the injury is performed, the incision is cauterized without ligating either the femoral artery or muscular branch. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52403/52403fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<img alt="Figure 2" src="/files/ftp_upload/52403/52403fig2highres.jpg" /> 
Figure 2: Higher magnification photographs of the critical steps of the two techniques for wire injury. Close-up comparison of steps for the ligation method (top) to the cautery method (bottom). The location of the arteriotomy is demarcated with the dashed line. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52403/52403fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<img alt="Figure 3" src="/files/ftp_upload/52403/52403fig3highres.jpg" /> 
Figure 3: Representative histological results of wire injury 14 days after the procedure. Histological from the arteries sections were stained with Hematoxylin and Eosin (top) and Movat&#8217;s pentachrome staining (bottom). Labeled in the image are the lumen (L), intima (I) and elastic lamina (EL).

Watch this Scientific Journal Video about Murine Model of Femoral Artery Wire Injury with Implantation of a Perivascular Drug Delivery Patch at JoVE.com

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

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. ...

murine-model-femoral-artery-wire-injury-with-implantation

Nanyang Technological University

Research

JoVE Journal

Medicine

17.9K Views.  University of Texas at Austin. 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. 

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

Programming Stem Cells for Therapeutic Angiogenesis Using Biodegradable Polymeric Nanoparticles

Controlled vascular growth is critical for successful tissue regeneration and wound healing, as well as for treating ischemic diseases such as stroke, heart attack or peripheral arterial diseases. Direct delivery of angiogenic growth factors has the potential to stimulate new blood vessel growth, but is often associated with limitations such as lack of targeting and short half-life in vivo. Gene therapy offers an alternative approach by delivering genes encoding angiogenic factors, but often requires using virus, and is limited by safety concerns. Here we describe a recently developed strategy for stimulating vascular growth by programming stem cells to overexpress angiogenic factors in situ using biodegradable polymeric nanoparticles. Specifically our strategy utilized stem cells as delivery vehicles by taking advantage of their ability to migrate toward ischemic tissues in vivo. Using the optimized polymeric vectors, adipose-derived stem cells were modified to overexpress an angiogenic gene encoding vascular endothelial growth factor (VEGF). We described the processes for polymer synthesis, nanoparticle formation, transfecting stem cells in vitro, as well as methods for validating the efficacy of VEGF-expressing stem cells for promoting angiogenesis in a murine hindlimb ischemia model.

We describe the method of programming stem cells to overexpress therapeutic factors for angiogenesis using biodegradable polymeric nanoparticles. Processes described include polymer synthesis, transfecting adipose-derived stem cells in vitro, and validating the efficacy of programmed stem cells to promote angiogenesis in a murine hindlimb ischemia model.

Controlled vascular growth is critical for successful tissue regeneration and wound healing, as well as for treating ischemic diseases such as stroke, ...

Bioengineering

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 ...

Osmotic Drug Delivery to Ischemic Hindlimbs and Perfusion of Vasculature with Microfil for Micro-Computed Tomography Imaging

Preclinical research in animal models of peripheral arterial disease plays a vital role in testing the efficacy of therapeutic agents designed to stimulate microcirculation. The choice of delivery method for these agents is important because the route of administration profoundly affects the bioactivity and efficacy of these agents1,2. In this article, we demonstrate how to locally administer a substance in ischemic hindlimbs by using a catheterized osmotic pump. This pump can deliver a fixed volume of aqueous solution continuously for an allotted period of time. We also present our mouse model of unilateral hindlimb ischemia induced by ligation of the common femoral artery proximal to the origin of profunda femoris and epigastrica arteries in the left hindlimb. Lastly, we describe the in vivo cannulation and ligation of the infrarenal abdominal aorta and perfusion of the hindlimb vasculature with Microfil, a silicone radiopaque casting agent. Microfil can perfuse and fill the entire vascular bed (arterial and venous), and because we have ligated the major vascular conduit for exit, the agent can be retained in the vasculature for future ex vivo imaging with the use of small specimen micro-CT3.

We show here the in vivo insertion of an osmotic pump for constant local drug delivery and the creation of hindlimb ischemia in a mouse model. Moreover, the hindlimb vasculature is perfused with Microfil, a silicone radiopaque agent, to prepare for micro-computed tomography (micro-CT) imaging.

Preclinical research in animal models of peripheral arterial disease plays a vital role in testing the efficacy of therapeutic agents designed to ...

A Radio-telemetric System to Monitor Cardiovascular Function in Rats with Spinal Cord Transection and Embryonic Neural Stem Cell Grafts

High thoracic or cervical spinal cord injury (SCI) can lead to cardiovascular dysfunction. To monitor cardiovascular parameters, we implanted a catheter connected to a radio transmitter into the femoral artery of rats that underwent a T4 spinal cord transection with or without grafting of embryonic brainstem-derived neural stem cells expressing green fluorescent protein. Compared to other methods such as cannula insertion or tail-cuff, telemetry is advantageous to continuously monitor blood pressure and heart rate in freely moving animals. It is also capable of long term multiple data acquisitions. In spinal cord injured rats, basal cardiovascular data under unrestrained condition and autonomic dysreflexia in response to colorectal distension were successfully recorded. In addition, cardiovascular parameters before and after SCI can be compared in the same rat if a transmitter is implanted before a spinal cord transection. One limitation of the described telemetry procedure is that implantation in the femoral artery may influence the blood supply to the ipsilateral hindlimb.

We present a protocol for using a radio-telemetric system to record cardiovascular parameters in T4 spinal cord transected rats eight weeks after embryonic brainstem neural stem cell grafting into the lesion site. Telemetry is an advanced technique to accurately evaluate cardiovascular function in conscious freely moving spinal cord injured rats.

High thoracic or cervical spinal cord injury (SCI) can lead to cardiovascular dysfunction. To monitor cardiovascular parameters, we implanted a catheter ...

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 ...

A Hydrogel Construct and Fibrin-based Glue Approach to Deliver Therapeutics in a Murine Myocardial Infarction Model.

The murine MI model is widely recognized in the field of cardiovascular disease, and has consistently been used as a first step to test the efficacy of treatments in vivo1. The traditional, established protocol has been further fine-tuned to minimize the damage to the animal. Notably, the pectoral muscle layers are teased away rather than simply cut, and the thoracotomy is approached intercostally as opposed to breaking the ribs in a sternotomy, preserving the integrity of the ribcage. With these changes, the overall stress on the animal is decreased. 
Stem cell therapies aimed to alleviate the damage caused by MIs have shown promise over the years for their pro-angiogenic and anti-apoptotic benefits. Current approaches of delivering cells to the heart surface typically involve the injection of the cells either near the damaged site, within a coronary artery, or into the peripheral blood stream2-4. While the cells have proven to home to the damaged myocardium, functionality is limited by their poor engraftment at the site of injury, resulting in diffusion into the blood stream5. This manuscript highlights a procedure that overcomes this obstacle with the use of a cell-encapsulated hydrogel patch. The patch is fabricated prior to the surgical procedure and is placed on the injured myocardium immediately following the occlusion of the left coronary artery. To adhere the patch in place, biocompatible external fibrin glue is placed directly on top of the patch, allowing for it to dry to both the patch and the heart surface. This approach provides a novel adhesion method for the application of a delicate cell-encapsulating therapeutic construct.

This protocol aims to alleviate the limitation of poor cell engraftment for stem cell treatment of myocardial infarctions through the use of a hydrogel system and a fibrin-based glue. With this approach, cell-to-tissue contact post-infarction can be maintained, increasing the therapeutic potential of beneficial agents at the site of injury.

The murine MI model is widely recognized in the field of cardiovascular disease, and has consistently been used as a first step to test the efficacy of ...

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. ...

Patch Angioplasty in the Rat Aorta or Inferior Vena Cava

Pericardial patches are commonly used in vascular surgery to close vessels. To facilitate studies of the neointimal hyperplasia that forms on the patch, we developed a rat model of patch angioplasty that can be used in either a vein or an artery, creating a patch venoplasty or a patch arterioplasty, respectively. Technical aspects of this model are discussed. The infra-renal IVC or aorta are dissected and then clamped proximally and distally. A 3 mm venotomy or arteriotomy is performed in the infrarenal inferior vena cava or aorta of 6 to 8 week-old Wistar rats. A bovine pericardial patch (3 mm x 1.5 mm x 0.6 mm) is then used to close the site using a 10-0 nylon suture. Compared to arterial patches, venous patches show increased neointimal thickness on postoperative day 7. This novel model of pericardial patch angioplasty can be used to examine neointimal hyperplasia on vascular biomaterials, as well as to compare the differences between the arterial and venous environments.

We have established a model of pericardial patch angioplasty that can be used in either small-diameter veins or arteries. This model can be used to compare venous and arterial neointimal hyperplasia formation.

Pericardial patches are commonly used in vascular surgery to close vessels. To facilitate studies of the neointimal hyperplasia that forms on the patch, ...

Transplantation of a 3D Bioprinted Patch in a Murine Model of Myocardial Infarction

Testing regenerative properties of 3D bioprinted cardiac patches in vivo using murine models of heart failure via permanent left anterior descending (LAD) ligation is a challenging procedure and has a high mortality rate due to its nature. We developed a method to consistently transplant bioprinted patches of cells and hydrogels onto the epicardium of an infarcted mouse heart to test their regenerative properties in a robust and feasible way. First, a deeply anesthetized mouse is carefully intubated and ventilated. Following left lateral thoracotomy (surgical opening of the chest), the exposed LAD is permanently ligated and the bioprinted patch transplanted onto the epicardium. The mouse quickly recovers from the procedure after chest closure. The advantages of this robust and quick approach include a predicted 28-day mortality rate of up to 30% (lower than the 44% reported by other studies using a similar model of permanent LAD ligation in mice). Moreover, the approach described in this protocol is versatile and could be adapted to test bioprinted patches using different cell types or hydrogels where high numbers of animals are needed to optimally power studies. Overall, we present this as an advantageous approach which may change preclinical testing in future studies for the field of cardiac regeneration and tissue engineering.

This protocol aims to transplant a 3D bioprinted patch onto the epicardium of infarcted mice modeling heart failure. It includes details regarding anesthesia, the surgical chest opening, permanent ligation of the left anterior descending (LAD) coronary artery and application of a bioprinted patch onto the infarcted area of the heart.

Testing regenerative properties of 3D bioprinted cardiac patches in vivo using murine models of heart failure via permanent left anterior descending (LAD) ...

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 ...