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...

<ol>
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P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Murine+model+of+hindlimb+ischemia.">Murine model of hindlimb ischemia.</a> J Vis Exp. , (2009).</li><li>McClung, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skeletal+muscle-specific+genetic+determinants+contribute+to+the+differential+strain-dependent+effects+of+hindlimb+ischemia+in+mice.">Skeletal muscle-specific genetic determinants contribute to the differential strain-dependent effects of hindlimb ischemia in mice.</a> Am. J. Pathol. 180, 2156-2169 (2012).</li><li>Dokun, A. O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+quantitative+trait+locus+(LSq-1)+on+mouse+chromosome+7+is+linked+to+the+absence+of+tissue+loss+after+surgical+hindlimb+ischemia.">A quantitative trait locus (LSq-1) on mouse chromosome 7 is linked to the absence of tissue loss after surgical hindlimb ischemia.</a> Circulation. 117, 1207-1215 (2008).</li><li>Rivard, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-dependent+impairment+of+angiogenesis.">Age-dependent impairment of angiogenesis.</a> Circulation. 99, 111-120 (1999).</li><li>Hazarika, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=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.">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.</a> Circ. 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A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Variations+in+surgical+procedures+for+hind+limb+ischaemia+mouse+models+result+in+differences+in+collateral+formation.">Variations in surgical procedures for hind limb ischaemia mouse models result in differences in collateral formation.</a> Eur. J. Vasc. Endovasc. Surg. 40, 796-803 (2010).</li><li>Kochi, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+the+arterial+anatomy+of+the+murine+hindlimb:+functional+role+in+the+design+and+understanding+of+ischemia+models.">Characterization of the arterial anatomy of the murine hindlimb: functional role in the design and understanding of ischemia models.</a> PloS one. 8, e84047 (2013).</li></ol>

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 ...

The overall goal of this protocol is to model human peripheral artery disease by the placement of sutures or ameroid constrictors on the mouse femoral artery for the induction of acute or subacute hindlimb ischemia, respectively. Peripheral artery disease is an important clinical problem, and these techniques using animal models have been developed to better represent the human disease. The primary advantage of these techniques is that the severity and timing of the onset of limb ischemia can be controlled by the investigator.Demonstrating the procedure will be Mike Padgett, a technician from my laboratory. One to two days before the procedure, confirm that the mouse is fully sedated by lack of response to toe pinch, and apply eye ointment. Next, use a small electric trimmer to shave the hair from both hindlimbs.When all of the hair has been trimmed, apply pre-warmed hair removal cream. After one minute, gently wipe away the cream with a moistened gauze pad. Then place the mouse in an empty paper towel lined recovery cage until it is able to maintain sternal recumbency.On the day of the surgery, place the fully sedated mouse under a technic stereo dissection microscope in the supine position, and secure both legs with surgical tape. If a temperature controlled heating pad is used, attach the temperature probe, and secure the probe to the base of the surgical platform with surgical tape. Next, use three alternating povidone iodine and alcohol wipes to clean the surgical site, and cover the animal with a sterile surgical drape with a hole at the incision site.Using a scalpel, make an initial incision along the center of the medial thigh from the knee toward the abdomen. Then use fine scissors to lengthen the incision to approximately one centimeter. Using forceps, open the incision to expose the membrane covering the inguinal fat tissue.Then use closed forceps to pierce through the membrane, into the space between the fat tissue and the abdomen. Gently release the pressure on the forceps to separate the fat tissue from the abdominal muscles, exposing the neurovascular bundle. It is crucial that the major blood vessels and their branches are properly identified prior to the dissection of the neurovascular bundle.Variations in the location of the constrictors or the sites of ligation can significantly alter the severity of the ischemia, and lead to a substantial variability in the outcomes. Insert a retractor into the incision, and pull the abdominal tissue toward the animal's head to expose the proximal ameroid constrictor site, which is just proximal to the lateral circumflex femoral artery. Then insert one medial and one lateral retractor into the distal part of the incision to widen the surgical field, pulling the inguinal fat tissue toward the foot and away from the surgical site.The proximal and superficial caudal branches of the femoral artery should be easily observed. The lateral circumflex femoral artery is located about five millimeters proximal to the caudal branches, and may be obscured by membrane. Now gently slide the tip of one forceps under the membrane that binds the vessels together to insert 1/2 of the tip between the vein and the artery.Then closet he forceps and gently tear away the membrane. When all of the membrane has been removed, insert the tip of the closed forceps between the vein and the artery, and release the pressure on the forceps to create a gap between the vessels. For subacute limb ischemia, slide the tip of the forceps under the femoral artery to isolate the vessel from the neurovascular bundle.Use a second set of angled forceps to grip the edge of the constrictor, and guide the constrictor under the femoral artery. Then lay the artery into the constrictor slot. Next, position the distal constrictor on the femoral artery immediately proximal to where the artery bifurcates into the popliteal and saphenous arteries, and place the femoral artery into the constrictor slot.For acute limb ischemia, thread a 7-0 suture under the artery at the same position as the proximal constrictor, and ligate the artery. Then tie a second ligature about one millimeter distal to the first, and use spring scissors to transect the artery between the two ligatures. For the distal arterial transection, place the ligatures about one millimeter apart, just proximal to where the femoral artery bifurcates into the popliteal and saphenous arteries.Then close the incision with interrupted five-0 vicryl sutures, and move the mouse to a 37 degree Celsius heating pad beneath a laser doppler perfusion imager. Now turn on the imager, and launch the image capture software. Click the New Single Image icon to open the scanner setup window, and set the scan size to large, and the scan speed to four milliseconds per pixel.Change the X and Y values under the Scan Area units panel to the appropriate values, then click the Video and Distance tab, and arrange both of the hindlimbs within the scan area. Click Auto Distance, then click Next to open the Subject Details window, and enter the subject information. Click Next to move on to the scanning window.Then click the Start Measurement button to open the Confirm or Override Scan Distance dialog box, and click OK to begin the scanning process. When the scanning is complete, move the mouse to an empty recovery cage with monitoring until it is fully recovered. In addition to the variations inherent in animal studies, other factors can introduce variability within the laser doppler perfusion imaging, including the body temperature, and the position of the animal.The subacute hindlimb ischemia model is dependent on the quality of the ameroid constrictors, which can vary widely within a given batch. Laser doppler perfusion imaging should be performed immediately after the procedure while the animal is still anesthetized to demonstrate the effects of the surgery, and to establish a baseline post-operative level of perfusion. A complete loss of hindlimb perfusion should be observed after the induction of acute limb ischemia, whereas only a mild decrease in perfusion is typically observed after the ameroid constrictor placement in the subacute model.These procedures allow exploration of the effects of varying degrees of ischemia on vascular remodeling and skeletal muscle regeneration in the mouse as a model of human peripheral artery disease. Once mastered, these procedures can be completed in about 15 minutes if they are performed correctly. While attempting these surgeries, it is important to remember to continually monitor the body temperature and the adequacy of the anesthesia in the animal.By adjusting the placement of the ligatures or the constrictors, the severity of the ischemic injury can be modified, which can be an important variable depending on the animal's genetic background, or the presence of cardiovascular risk factors. After watching this video, you should have a good understanding of how to induce both acute and subacute hindlimb ischemia in the mouse, and to define the vascular anatomy to ensure a proper outcome and reproducibility of the procedure.

methods-for-acute-and-subacute-murine-hindlimb-ischemia

Research

JoVE Journal

Medicine

16.0K visualizações.  Duke University Medical Center. The overall goal of this protocol is to model human peripheral artery disease by the placement of sutures or ameroid constrictors on the mouse femoral artery for the induction of acute or subacute hindlimb ischemia, respectively. Peripheral artery disease is an important clinical problem, and these techniques using animal models have been developed to better represent the human disease. The primary advantage of these techniques is that the severity and timing of the onset of limb ischemia can...