Methods for Acute and Subacute Murine Hindlimb Ischemia

Summary

Surgical induction of hindlimb ischemia in the mouse is useful to examine angiogenesis, however this is compromised in certain inbred mouse strains that display marked ischemia-induced tissue necrosis. Methods are described to induce subacute limb ischemia using ameroid constrictors to circumvent this problem through the induction of gradual arterial occlusion.

Abstract

Peripheral artery disease (PAD) is a leading cause of cardiovascular morbidity and mortality in developed countries, and animal models that reliably reproduce the human disease are necessary to develop new therapies for this disease. The mouse hindlimb ischemia model has been widely used for this purpose, but the standard practice of inducing acute limb ischemia by ligation of the femoral artery can result in substantial tissue necrosis, compromising investigators' ability to study the vascular and skeletal muscle tissue responses to ischemia. An alternative approach to femoral artery ligation is the induction of gradual femoral artery occlusion through the use of ameroid constrictors. When placed around the femoral artery in the same or different locations as the sites of femoral artery ligation, these devices occlude the artery over 1 - 3 days, resulting in more gradual, subacute ischemia. This results in less substantial skeletal muscle tissue necrosis, which may more closely mimic the responses seen in human PAD. Because genetic background influences outcomes in both the acute and subacute ischemia models, consideration of the mouse strain being studied is important in choosing the best model. This paper describes the proper procedure and anatomical placement of ligatures or ameroid constrictors on the mouse femoral artery to induce subacute or acute hindlimb ischemia in the mouse.

Introduction

Peripheral artery disease (PAD) is a leading cause of cardiovascular morbidity and mortality in developed countries ¹. PAD results from atherosclerotic obstruction of the peripheral arteries that leads to limb ischemia with resultant exertional or rest pain and occasionally non-healing ulcers and gangrene that necessitate limb amputation. Therapies targeting PAD are directed primarily towards endovascular ² or surgical revascularization ³, as essentially no effective medical therapies exist ⁴.

Unfortunately, revascularization is often of limited benefit, as bypass grafts have high failure rates (u....

Protocol

All animal experiments were performed according to protocol approved by the Duke Institutional Animal Care and Use Committee. Male mice were used in this study, although either sex can be used as indicated for the scientific purpose of the study.

1. Hair Removal

1. Prior to induction of anesthesia, set up a pre-surgical preparation area consisting of a covered heating pad set at 37 °C and a nosecone port connected to continuous flow of isoflurane.
2. Place the mouse in the anesthesia induction chamber. Set the O₂ flow meter to 1 L/min and isoflurane to 1 - 3%.
   NOTE: Anesthesia is typically in....

Results

Proper identification of the mouse hindlimb vasculature is critical to ensuring reproducibility of the techniques for inducing both subacute and acute hindlimb ischemia, as described here. In addition to the variation inherent in animal studies, other factors can introduce variability in laser Doppler perfusion imaging (LDPI), including the type of anesthesia, position of the animal (supine vs. prone), and body temperature (see Figure 3). In addition, the subacut.......

Discussion

Perhaps the most challenging step in this procedure is the separation of the femoral artery from the femoral vein. The larger diameter and thinner walls of the femoral vein compared to those of the artery increase its susceptibility to puncture and tearing during surgical manipulation. The likelihood of disrupting the vein can be reduced by keeping the wound moist using a sterile swab moistened with PBS. It is also important to ensure that all forceps are sharpened, aligned, and free of breaks in order to allow precise m.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Dumont #5/45 Forceps	Fine Science Tools	11251-35	Dumoxel
Dumont Style 5 Mini Forceps	Fine Science Tools	11200-14	Inox
Extra Fine Bonn Scissors	Fine Science Tools	14084-08
7 - 0 Silk Suture	Sharpoint	DA-2527N
5 - 0 Coated Vicryl Suture	Ethicon	J463G
Graefe Forceps	Fine Science Tools	11053-10
Vannas Spring Scissors	Fine Science Tools	15000-03
Artifical Tears Ointment	Rugby Laboratories	0536-6550-91
Surgical Tape	3M	1530-0
Fine Cotton Swabs	Contec	SC-4
Temperature Controller	Physitemp	TCAT-2DF
Ameroid Constrictors	Research Instruments SW	MMC-0.25 x 1.00-SS
Hot Bead Sterilizer
Deltaphase Isothermal Pad	Braintree Scientific	39DP
Needle Driver	Fine Science Tools
Phosphate Buffered Saline	Gibco	10010-023
Moor LDPI	Moor Instruments	moorLDI2
moorLDI Measurement software	Moor Instruments	v. 6.0
Hair Removal Cream	Nair