Name: methods for acute and subacute murine hindlimb ischemia
Uploaded: 2016-06-21T15:00:00
Description: Watch this Scientific Journal Video about Methods for Acute and Subacute Murine Hindlimb Ischemia at JoVE.com

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

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
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	<li>Prior to induction of anesthesia, set up a pre-surgical preparation area consisting of a covered heating pad set at 37 &#176;C and a nosecone port connected to continuous flow of isoflurane.</li>
	<li>Place the mouse in the...

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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...

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 (up to 50% within 5 years) 5 that are worse in some populations (e.g., smokers, women, non-saphenous vein grafts) 6,7. Endovascular approaches, such as angioplasty and stenting, are also compromised by high restenosis rates (in excess of 50% within 1 year), particulary in femoropopliteal disease 8, although the use of drug-eluting balloons and stents has improved outcomes somewhat 9-11. In order to develop new treatments for PAD it is essential to develop animal models that reliably reproduce the human disease.
To date, the most common model of PAD is the hindlimb ischemia model (HLI), which is most frequently performed in mice 12,13. In its most common manifestation, the model entails surgical ligation of the proximal and distal femoral artery and its intervening side-branches followed by excision of the vessel, resulting in occlusion of blood flow and induction of acute limb ischemia. HLI has been used primarily to study the angiogenic and arteriogenic responses in peripheral limb muscle tissue and the effects of various therapies (e.g., drugs, gene delivery, stem cells) on these responses. More recently, our group has used this model to examine the role of skeletal muscle cells in the response to limb ischemia and the effects of genetic differences on outcomes 14.
The HLI model has facilitated our current understanding that the vascular and muscle responses to ischemia are dependent on genetics (i.e., inbred strain) 15, age 16, and the presence or absence of other diseases or conditions relevant to atherosclerosis, including diabetes mellitus 17 and hypercholesterolemia 18. However, an important weakness of the traditional HLI model is that it is a model of acute limb ischemia 12,13, whereas human PAD causes chronic ischemia as a result of the gradual development of occlusive atherosclerotic lesions in the peripheral arteries.
In an attempt to circumvent this weakness, Tang and colleagues initially developed a rat model of gradual femoral arterial occlusion using ameroid constrictors 19, and the same group subsequently developed a similar mouse model 20. Ameroid constrictors were described initially in the 1950s in a canine model of chronic myocardial ischemia 21,22. These devices have an outer metal sleeve encasing an inner layer of a hygroscopic material, usually casein, and when placed around an artery they induce gradual vessel occlusion as they absorb moisture from the surrounding tissues. In their modification of the model, Yang et al. placed constrictors on both the proximal and distal femoral artery at sites analogous to the surgical ligation sites, and they ligated the side branches of the femoral artery, as in the traditional model. Compared to acute HLI, ameroid constrictor-induced ischemia resulted in lower expression of inflammatory and shear stress-dependent genes, lower blood flow recovery 4 - 5 weeks post-operatively, and less muscle necrosis 20. Based on these observations, it was felt that gradual arterial occlusion might provide a model of PAD more relevant to the human disease.
Notably, in the original report, effects of ameroid constrictor-induced ischemia were examined only in C57BL/6 mice 19, which are relatively resistant to ischemia-induced muscle necrosis 15. We recently modified the gradual ischemia model further and explored its effects in the more ischemia-susceptible BALB/c mouse strain 23. In the first manifestation of the model, we placed constrictors on both the proximal and distal femoral artery but left all side-branches intact. In a second, milder modification, we placed a single constrictor only on the proximal femoral artery and again left all side-braches of the artery intact. In both modifications of this model, we found that BALB/c mice, but not C57BL/6 mice, displayed significant muscle necrosis despite having similar blood flow and vascular density. Similar to our previous study 14, these findings demonstrated that limb muscle injury is not solely influenced by blood flow, but is in part dependent on genetic background. Moreover, we found that limb blood flow fell to its nadir within 3 days, thus the model appears to be more one of &#39;subacute&#39; rather than gradual limb ischemia.
Based on these prior studies, it appears clear that a single method for inducing hindlimb ischemia may not be suitable in all cases. Because a variety of conditions (e.g., genetic differences and presence or absence of co-morbid conditions) influence both the vascular and skeletal muscle-specific responses, investigators may find it necessary to modify the chronicity and/or the severity of hindlimb ischemia to best suit their purposes. Furthermore, prior descriptions of the model typically lacked suitable anatomical landmarks to facilitate reliable inter-investigator reproducibility of the technique. In this paper, methods for inducing either acute or subacute hindlimb ischemia in the mouse are described, and precise anatomical landmarks are provided.

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methods for acute and subacute murine hindlimb ischemia

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&#39; 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.

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...

Watch this Scientific Journal Video about Methods for Acute and Subacute Murine Hindlimb Ischemia at JoVE.com

Methods for Acute and Subacute Murine Hindlimb Ischemia

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 ...

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