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. 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.
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
NOTE: All methods shown in this protocol have been approved by the Institutional Animal Care and Use Committee.
1. Preparation of Surgical Table
2. Preparation of Mouse for Surgery
3. Isolation of the Femoral Artery
4. Performance of Femoral Artery Wire Injury
5. Ligating the Muscular Branch
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.
7. Implantation of Perivascular Drug Delivery Patch
8. Wound Closure and Recovery
9. Harvesting Femoral Arteries for Histology
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’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.
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. Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
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’s pentachrome staining (bottom). Labeled in the image are the lumen (L), intima (I) and elastic lamina (EL).
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.
The authors have nothing to disclose.
The authors would like to acknowledge support through the American Heart Association (10SDG2630139), the Welch Foundation and through the NIH Director’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.
Name | Company | Catalog Number | Comments |
Straight spring wire, 0.15” diameter | Cook | G02426 | |
High Temperature cautery | Bovie Medical Corp. | HIT1 | |
High-temperature fine tip for cautery | Bovie Medical Corp. | H101 | |
Micro-scissors | Fine Science Tools | 15000-13 | For performance of arteriotomy |
Angled fine-tipped forceps | Fine Science Tools | 11251-35 | For blunt dissection of vascular bundle |
Angled forceps | Roboz | RS-5069 | For clearing tissues |
Surgical scissors | Roboz | RS-5840 | For cutting skin |
Retractor | Fine Science Tools | 18200-10 | |
Retractor wire | Fine Science Tools | 18200-05 | Attached to retractor |
Base plate | Fine Science Tools | 18200-03 | For use with retractor |
Magnetic retractor fixator | Fine Science Tools | 18200-01 | |
Needle Holder | Roboz | RS-7822 | |
Hemostatic forceps | Biomedical Research Instruments, Inc. | 34-1000 | |
Dissecting microscope | Meiji Techno | EMZ-5TR | |
Microscope light source | Meiji Techno | FT191 | |
Warm water recirculator | Gaymar | TP-500 | For maintaining mouse body temperature |
Reusable heating pad | Gaymar | TP-R 22G | For maintaining mouse body temperature |
Lidocaine | Various | ||
4.0 Vicryl suture with half circle needle | Ethicon | J494G | For post-surgical wound closure |
Sterile cotton-tipped applicators | Puritan | 25-806-2WC | For application of depilatory cream and absorbing fluids |
Depilatory cream | Nair | ||
Isoflurane | Various | ||
Betadine | Various | ||
70% ethanol | Various | ||
6.0 braided silk suture | Teleflex Medical | 4-S | For isolation of femoral artery during surgery |
0.9% sodium chloride | Various | For irrigating tissues | |
Gel eye lubricant | Various | ||
Glass petri dish | Pyrex | 3160-60 | For femoral artery harvest |
10% buffered formalin | Various | For fixation of femoral artery | |
70% ethanol | Various | For fixation of femoral artery | |
Bouin's fluid | Electron Microscopy Sciences | For Movat's Pentachrome staining | |
Alcian blue, 1% | Electron Microscopy Sciences | 26385-01 | For Movat's Pentachrome staining |
Alkaline alcohol | Electron Microscopy Sciences | 26385-02 | For Movat's Pentachrome staining |
Orcein, 0.2% | Electron Microscopy Sciences | 26385-03 | For Movat's Pentachrome staining |
Hematoxylin alcoholic, 5% | Electron Microscopy Sciences | 26385-04 | For Movat's Pentachrome staining |
Ferric chloride, 10% | Electron Microscopy Sciences | 26385-05 | For Movat's Pentachrome staining |
Lugol's Iodine | Electron Microscopy Sciences | 26385-06 | For Movat's Pentachrome staining |
Woodstain scarlet-acid fuchsin working solution | Electron Microscopy Sciences | 26385-07 | For Movat's Pentachrome staining |
Acetic acid, 0.5% | Electron Microscopy Sciences | Various | For Movat's Pentachrome staining |
Phosphotungstic acid, 5% | Electron Microscopy Sciences | 26385-09 | For Movat's Pentachrome staining |
Alcoholic saffron, 6% | Electron Microscopy Sciences | 26385-10 | For Movat's Pentachrome staining |