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Medicine

Methods for Acute and Subacute Murine Hindlimb Ischemia

Published: June 21st, 2016

DOI:

10.3791/54166

1Division of Cardiology, Department of Medicine, Duke University Medical Center

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.

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.

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....

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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.......

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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.......

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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.......

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This study was supported by NIH grants R21HL118661, R56HL124444, and R01HL124444 to CDK, and by NIH grants R00HL103797 and R01HL125695 to JMM.

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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

  1. Criqui, M. H., Aboyans, V. Epidemiology of peripheral artery disease. Circ. Res. 116, 1509-1526 (2015).
  2. Thukkani, A. K., Kinlay, S. Endovascular intervention for peripheral artery disease. Circ. Res. 116, 1599-1613 (2015).
  3. Vartanian, S. M., Conte, M. S. Surgical intervention for peripheral arterial disease. Circ. Res. 116, 1614-1628 (2015).
  4. Bonaca, M. P., Creager, M. A. Pharmacological treatment and current management of peripheral artery disease. Circ. Res. 116, 1579-1598 (2015).
  5. Conte, M. S., et al. Design and rationale of the PREVENT III clinical trial: edifoligide for the prevention of infrainguinal vein graft failure. Vasc Endovascular Surg. 39, 15-23 (2005).
  6. Pomposelli, F. B., et al. A decade of experience with dorsalis pedis artery bypass: analysis of outcome in more than 1000 cases. J. Vasc. Surg. 37, 307-315 (2003).
  7. Willigendael, E. M., et al. Smoking and the patency of lower extremity bypass grafts: a meta-analysis. J. Vasc. Surg. 42, 67-74 (2005).
  8. Schillinger, M., et al. Balloon angioplasty versus implantation of nitinol stents in the superficial femoral artery. N. Engl. J. Med. 354, 1879-1888 (2006).
  9. Marmagkiolis, K., et al. 12-month primary patency rates of contemporary endovascular device therapy for femoro-popliteal occlusive disease in 6,024 patients: beyond balloon angioplasty. Catheter. Cardiovasc. Interv. 84, 555-564 (2014).
  10. Rosenfield, K., et al. Trial of a Paclitaxel-Coated Balloon for Femoropopliteal Artery Disease. N. Engl. J. Med. 373, 145-153 (2015).
  11. Tepe, G., et al. Drug-coated balloon versus standard percutaneous transluminal angioplasty for the treatment of superficial femoral and popliteal peripheral artery disease: 12-month results from the IN.PACT SFA randomized trial. Circulation. 131, 495-502 (2015).
  12. Couffinhal, T., et al. Mouse model of angiogenesis. Am. J. Pathol. 152, 1667-1679 (1998).
  13. Niiyama, H., Huang, N. F., Rollins, M. D., Cooke, J. P. Murine model of hindlimb ischemia. J Vis Exp. , (2009).
  14. McClung, J. M., et al. Skeletal muscle-specific genetic determinants contribute to the differential strain-dependent effects of hindlimb ischemia in mice. Am. J. Pathol. 180, 2156-2169 (2012).
  15. Dokun, A. O., et al. A quantitative trait locus (LSq-1) on mouse chromosome 7 is linked to the absence of tissue loss after surgical hindlimb ischemia. Circulation. 117, 1207-1215 (2008).
  16. Rivard, A., et al. Age-dependent impairment of angiogenesis. Circulation. 99, 111-120 (1999).
  17. Hazarika, S., et al. Impaired angiogenesis after hindlimb ischemia in type 2 diabetes mellitus: differential regulation of vascular endothelial growth factor receptor 1 and soluble vascular endothelial growth factor receptor 1. Circ. Res. 101, 948-956 (2007).
  18. Couffinhal, T., et al. Impaired collateral vessel development associated with reduced expression of vascular endothelial growth factor in ApoE-/- mice. Circulation. 99, 3188-3198 (1999).
  19. Tang, G. L., Chang, D. S., Sarkar, R., Wang, R., Messina, L. M. The effect of gradual or acute arterial occlusion on skeletal muscle blood flow, arteriogenesis, and inflammation in rat hindlimb ischemia. J. Vasc. Surg. 41, 312-320 (2005).
  20. Yang, Y., et al. Cellular and molecular mechanism regulating blood flow recovery in acute versus gradual femoral artery occlusion are distinct in the mouse. J. Vasc. Surg. 48, 1546-1558 (2008).
  21. Litvak, J., Siderides, L. E., Vineberg, A. M. The experimental production of coronary artery insufficiency and occlusion. Am. Heart J. 53, 505-518 (1957).
  22. Bredee, J. J. An improved ameroid constrictor. Preliminary communication. J. Surg. Res. 9, 107-112 (1969).
  23. McClung, J. M., et al. Subacute limb ischemia induces skeletal muscle injury in genetically susceptible mice independent of vascular density. J. Vasc. Surg. , (2015).
  24. Hellingman, A. A., et al. Variations in surgical procedures for hind limb ischaemia mouse models result in differences in collateral formation. Eur. J. Vasc. Endovasc. Surg. 40, 796-803 (2010).
  25. Kochi, T., et al. Characterization of the arterial anatomy of the murine hindlimb: functional role in the design and understanding of ischemia models. PloS one. 8, e84047 (2013).

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