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In the United States, peripheral arterial disease (PAD) affects about 10 million individuals, and is also prevalent worldwide. Medical therapies for symptomatic relief are limited. Surgical or endovascular interventions are useful for some individuals, but long-term results are often disappointing. As a result, there is a need for developing new therapies to treat PAD. The murine hindlimb ischemia preparation is a model of PAD, and is useful for testing new therapies. When compared to other models of tissue ischemia such as coronary or cerebral artery ligation, femoral artery ligation provides for a simpler model of ischemic tissue. Other advantages of this model are the ease of access to the femoral artery and low mortality rate.
In this video, we demonstrate the methodology for the murine model of unilateral hindimb ischemia. The specific materials and procedures for creating and evaluating the model will be described, including the assessment of limb perfusion by laser Doppler imaging. This protocol can also be utilized for the transplantation and non-invasive tracking of cells, which is demonstrated by Huang et al.1.
The anatomy of the hindlimb vasculature is shown in Figure 12. A representative diagram of the hindlimb after femoral artery explantation is shown in Figure 2. To confirm the induction of ischemia to the hindlimb, laser Doppler perfusion image analysis demonstrates a dramatic reduction in blood flow to the ischemic limb, in comparison to the control limb, as shown in Figure 3.
Figure 1. Anatomy of the hindlimb vasculature. Asterisks indicate the locations of ligation for the induction of hindlimb ischemia.
Figure 2. Representative diagram showing the anatomy of the hindlimb after ligation of the femoral artery at the proximal and distal sites after the removal of the femoral artery.
Figure 3. Laser Doppler images showing blood flow before and after the induction of ischemia to the left hindlimb (indicated by arrow).
There are some sources of variability to consider while planning and performing hindlimb ischemia models. First, the level of ischemia may vary according to the location of the ligation with respect to that of side branches. Therefore, for consistency of the model, the animals should undergo the same level of arterial ligation. Another source of variability in ischemic recovery is related to the age of the animals, with young animals (6–8 weeks old) having faster and more complete recovery rates than older animals (8–10 months old), as assessed by hemodynamic (i.e. laser Doppler perfusion) or functional (i.e. treadmill testing) measures. For studies in which one is assessing an angiogenic agent, it may be preferable to use older animals, because a greater difference between groups may be observed with a therapeutic intervention. Conversely, for studies in which one is assessing an anti-angiogenic factor, it may be preferable to use younger animals to maximize effect size3.
If performed correctly, there should be minimal complications such as bleeding, infection, or mortality. If bleeding occurs by accidental disruption of the femoral vein or other vessels, moderate pressure with a sterile cotton tipped applicator or gauze should be applied to the site of hemorrhage until the bleeding stops. Animals showing signs of infection should be treated with anti-infective agents. Animals exhibiting significant gangrene may need to beeuthanized. This complication is more common in older animals, and in some strains, such as BALB c mice4. In addition, the hindlimb ischemia model may cause mild to moderate pain. Therefore, analgesics such as buprenorphine or carprofen should be administered as needed for treatment of pain, according to recommendations of the IACUC.
In conclusion, we have demonstrated a simple and reproducible method for studying PAD in a murine model of hindlimb ischemia. The hindlimb ischemia model with laser Doppler imaging analysis is an excellent system for studying vascular pathologies and for assessing therapeutic candidates.
The authors thank Andrea Axtell, Satoshi Itoh, MD, Jeff Velotta, MD, Grant Hoyt, Robert C. Robbins, MD, Jin Yu, MD, Tim Doyle, PhD, and the Stanford Small Animal Imaging Core for technical assistance. The authors also thank A.M. Bickford, Inc. for support of veterinary equipment. This research was supported by research grants from the National Institutes of Health (R01 HL-75774, R01 CA098303, R21 HL085743, 1K12 HL087746), the California Tobacco Related Disease Research Program of the University of California (15IT-0257 and 1514RT-0169), and the California Institute for Regenerative Medicine (RS1-00183). N.H. is supported by a fellowship from the American Heart Association.