The authors gratefully acknowledge funding through the Department of Defense Congressionally Directed Research Program (DOD CDMRP; W81XWH-16-1-0582) to ABB and RS. The authors also acknowledge funding through the American Heart Association (17IRG33410888), the DOD CDMRP (W81XWH-16-1-0580) and the National Institutes of Health (1R21EB023551-01; 1R21EB024147-01A1; 1R01HL141761-01) to ABB.

Studies involving animals were performed with the approval of the University of Texas at Austin and the UTHealth Science Center at Houston Institutional Animal Care and Use Committee (IACUC), the Animal Care and Use Review Office (ACURO) of The United States Army Medical Research and Materiel Command Office of Research Protections, and in accordance with NIH guidelines for animal care.
1. Induction of diabetes and hyperlipidemia
<ol>
	<li>Transition the New Zealand rabbits (4&#8211;6 months ...

We have presented a preclinical model for inducing hind limb ischemia in rabbits with diabetes and hyperlipidemia. In many studies, there is ambiguity to the technique used to create hind limb ischemia in rabbits. In mice, the severity and recovery from hind limb ischemia is highly dependent on the location the ligation and technique used to induce ischemia. The significance of the technique presented in this work is that it allows for the consistent induction of ischemia that does not fully recover after 8 weeks in diab...

Peripheral arterial disease (PAD) is a common circulatory disorder in which the progression of atherosclerotic plaque formation leads to a narrowing of blood vessels in the limbs of the body. The recent increase in risk factors for atherosclerosis, including diabetes, obesity, and inactivity, has led to increasing prevalence of vascular disease1. Currently, it is estimated that 12%&#8211;20% of the general population over 60 years old has peripheral arterial disease2. A major consequence of peripheral arterial disease is the development of peripheral ischemia, most commonly found in the lower limbs. In severe cases, patients can develop critical limb ischemia, a state in which there is constant pain due to a lack of blood flow. Patients with critical limb ischemia have a 50% likelihood of having one limb amputated within one year of diagnosis. Furthermore, patients with diabetes have a higher incidence of peripheral arterial disease and poorer outcomes following interventions for revascularization3,4. Current therapies for peripheral ischemia include percutaneous interventions such as atherectomy and stenting or surgical bypass. However, for many patients these treatments only provide short-term benefits and many are not healthy enough for major surgical procedures. In this work, we describe a preclinical animal model for testing new treatments targeting peripheral vascular disease that incorporates the generation of peripheral ischemia in rabbits through surgical ligation in the context of the diabetic disease state.
The hind limb ischemia model in rabbits has been used as a physiological model for obstructive vascular disease and preclinical precursor to human studies for over half a century5,6. Rabbits are often a preferred species for studies on peripheral ischemia due to the developed musculature of the ankle and calf muscle, in contrast to common large animal models that are ungulates (animals with hooves). Several recent reviews have addressed the use of this model and others in modeling peripheral vascular disease in humans7,8. Similar models using hind limb ischemia in rabbits were used in preclinical studies of growth factors9,10,11,12,13,14,15,16,17,18,19,20, gene therapy21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44, and stem cells45,46,47,48,49,50,51for therapeutic neovascularization in the limbs. Unfortunately, the clinical trials that followed these successful animal studies did not show significant benefits for patients52.
One suggested explanation of the reason for this translational failure is that the condition of peripheral ischemia in human patients is one that includes resistance to angiogenic signals53,54,55,56,57,58,59. Several studies have shown defects in angiogenic signaling pathways in diabetes and hyperglycemia. Diabetes and hyperlipidemia lead to a loss of heparan sulfate proteoglycans and an increase in enzymes that cut heparan sulfate, presenting a potential mechanism for resistance to therapeutic angiogenesis/arteriogenesis with growth factors60,61. Thus, a key feature of a model for peripheral ischemia should include an aspect of therapeutic resistance so that therapies may be evaluated in the context of the disease state present in human patients.
In this work, we describe a rabbit model of peripheral ischemia through surgical ligation of the femoral arteries. A lead-in period with the induction of diabetes and hyperlipidemia is incorporated into the model. We compared this model to another model that incorporates a higher fat diet without diabetes and found that the model with diabetes and lower level of hyperlipidemia was more effective in reducing blood vessel growth. Our model combines advancements that have been used by separate groups, with the goal of providing a practical and standardized method to achieve consistent results in peripheral vascular disease research.

<table>
	<tbody>
		<tr>
			<td>0.9% Sodium Chloride</td>
			<td>Henry Schein Medical</td>
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			<td>1 mL Syringe</td>
			<td>VWR</td>
			<td>BD309628</td>
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			<td>10 mL Syringe</td>
			<td>VWR</td>
			<td>BD309695</td>
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			<td>10% Formalin</td>
			<td>Fisher-Scientific</td>
			<td>23-245684</td>
			<td></td>
		</tr>
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			<td>18G Needle</td>
			<td>VWR</td>
			<td>89219-294</td>
			<td></td>
		</tr>
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			<td>20G Needle</td>
			<td>VWR</td>
			<td>89219-340</td>
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			<td>25G Needle</td>
			<td>VWR</td>
			<td>89219-290</td>
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			<td>27G Needle</td>
			<td>VWR</td>
			<td>89219-288</td>
			<td></td>
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			<td>5 mL Syringe</td>
			<td>VWR</td>
			<td>BD309646</td>
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			<td>5% Dextrose</td>
			<td>Patterson Veterinary</td>
			<td>07-800-9689</td>
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			<td>Acepromazine</td>
			<td>Patterson Veterinary</td>
			<td>VEDC207</td>
			<td></td>
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			<td>Alfaxalone</td>
			<td>Patterson Veterinary</td>
			<td>07-891-6051</td>
			<td></td>
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			<td>Alginate</td>
			<td>Sigma-Aldrich</td>
			<td>PHR1471-1G</td>
			<td></td>
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			<td>Alloxan Monohydrate</td>
			<td>Sigma-Aldrich</td>
			<td>A7413</td>
			<td></td>
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			<td>Angiography Equipment</td>
			<td>Toshiba</td>
			<td>Infinix-i</td>
			<td></td>
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			<td>Angiography Injector</td>
			<td>Medrad</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Mouse Ab Alexa 594</td>
			<td>Thermo Fisher Scientific</td>
			<td>A-11032</td>
			<td>Secondary Antibody for IHC</td>
		</tr>
		<tr>
			<td>Anti-Rabbit Ab Alexa 488</td>
			<td>Thermo Fisher Scientific</td>
			<td>A-11008</td>
			<td>Secondary Antibody for IHC</td>
		</tr>
		<tr>
			<td>a-SMA Antibody</td>
			<td>Abcam</td>
			<td>ab5694</td>
			<td>Primary Antibody for IHC</td>
		</tr>
		<tr>
			<td>Baytril</td>
			<td>Bayer Animal Health</td>
			<td>724089904201</td>
			<td>Enrofloxacin</td>
		</tr>
		<tr>
			<td>Blood Chemistry Panel</td>
			<td>IDEXX</td>
			<td>2616</td>
			<td>Rabbit Panel</td>
		</tr>
		<tr>
			<td>Blood Pressure Cuff</td>
			<td>WelchAllyn</td>
			<td></td>
			<td>Flexiport Disposable BP Cuff-infant size 7</td>
		</tr>
		<tr>
			<td>Blood Pressure Monitor</td>
			<td>Vmed Technology</td>
			<td></td>
			<td>Vmed Vet-Dop2</td>
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		<tr>
			<td>Bupivacaine</td>
			<td>Henry Schein Medical</td>
			<td>6023287</td>
			<td></td>
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			<td>Buprenorphine</td>
			<td>Patterson Veterinary</td>
			<td>42023017905</td>
			<td></td>
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			<td>Buprenorphine SR</td>
			<td>ZooPharm</td>
			<td></td>
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			<td>Calcium Sulfate</td>
			<td>CB Minerals</td>
			<td></td>
			<td>Food and Pharmaceutical Grade USP and FCC</td>
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		<tr>
			<td>Chlorhexidine Scrub</td>
			<td>Patterson Veterinary</td>
			<td>07-888-4598</td>
			<td></td>
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		<tr>
			<td>Chloroform</td>
			<td>Fisher-Scientific</td>
			<td>C298-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Cholesterol</td>
			<td>Sigma-Aldrich</td>
			<td>C8503</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Thermo Fisher Scientific</td>
			<td>62248</td>
			<td></td>
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			<td>Ear Vein Catheter</td>
			<td>Patterson Veterinary</td>
			<td>SR-OX165</td>
			<td>Surflo IV catheters</td>
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		<tr>
			<td>Endotracheal tube</td>
			<td>Patterson Veterinary</td>
			<td></td>
			<td>Sheridan Brand, Depends on Rabbit Size</td>
		</tr>
		<tr>
			<td>Glucometer</td>
			<td>Amazon</td>
			<td>B001A67WH2</td>
			<td>Accu-Chek Aviva</td>
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		<tr>
			<td>Glucometer Test Strips</td>
			<td>McKesson Medical-Surgical</td>
			<td>788222</td>
			<td>Accu-Chek Aviva Plus</td>
		</tr>
		<tr>
			<td>Guidewire</td>
			<td>Boston Scientific</td>
			<td>39122-01</td>
			<td></td>
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			<td>Hair Clippers</td>
			<td>Amazon</td>
			<td>B000CQZI3Q</td>
			<td>Oster #40 blade</td>
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			<td>Heating Pad</td>
			<td>Cincinnati Subzero</td>
			<td>273</td>
			<td></td>
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			<td>Heating Pad Pump</td>
			<td>Gaymar</td>
			<td></td>
			<td>Gaymar T/Pump</td>
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			<td>Hemostat</td>
			<td>Fine Science Tools</td>
			<td>13009-12</td>
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			<td>Heparin</td>
			<td>Patterson Veterinary</td>
			<td></td>
			<td></td>
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		<tr>
			<td>Insertion Tool</td>
			<td>Merit Medical Systems</td>
			<td>MAP550</td>
			<td>metal wire insertion tool</td>
		</tr>
		<tr>
			<td>Insulin</td>
			<td>HPB Pharmacy</td>
			<td></td>
			<td>Novalin R &#38; Novalin N</td>
		</tr>
		<tr>
			<td>Insulin Syringes</td>
			<td>McKesson Medical-Surgical</td>
			<td>942674</td>
			<td></td>
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			<td>Introducer</td>
			<td>Cook Medical</td>
			<td>G28954</td>
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			<td>Henry Schein Medical</td>
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			<td>Patterson Veterinary</td>
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			<td>McKesson Medical-Surgical</td>
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			<td>Lidocaine</td>
			<td>McKesson Medical-Surgical</td>
			<td>239936</td>
			<td></td>
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			<td>Lidocaine/Prilocaine cream</td>
			<td>McKesson Medical-Surgical</td>
			<td>761240</td>
			<td></td>
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			<td>Ligaloop</td>
			<td>V. Mueller</td>
			<td>CH117 / CH116</td>
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			<td>Mazola Corn Oil</td>
			<td>Amazon</td>
			<td>B0049IIVCI</td>
			<td></td>
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			<td>Medrad Syringe</td>
			<td>McKesson Medical-Surgical</td>
			<td>346920</td>
			<td>150 mL</td>
		</tr>
		<tr>
			<td>Meloxicam</td>
			<td>Patterson Veterinary</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Metal ball sutures</td>
			<td>Ethicon-Johnson &#38; Johnson</td>
			<td>K891H</td>
			<td>4-0 silk C-1 30&#34;</td>
		</tr>
		<tr>
			<td>Metzenbaum Scissors</td>
			<td>Fine Science Tools</td>
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		<tr>
			<td>Midazolam</td>
			<td>Henry Schein Medical</td>
			<td>1215470</td>
			<td></td>
		</tr>
		<tr>
			<td>Nitroglycerin</td>
			<td>McKesson Medical-Surgical</td>
			<td>927528</td>
			<td></td>
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		<tr>
			<td>PECAM Antibody</td>
			<td>Novus Biologicals</td>
			<td>NB600-562</td>
			<td>Primary Antibody for IHC</td>
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		<tr>
			<td>Perfusion Pump</td>
			<td>Masterflex</td>
			<td></td>
			<td></td>
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			<td>Pigtail Catheter</td>
			<td>Merit Medical Systems</td>
			<td>1310-21-0053</td>
			<td>3F pigtail</td>
		</tr>
		<tr>
			<td>Polydioxanone (PDS II) suture</td>
			<td>McKesson Medical-Surgical</td>
			<td>129271</td>
			<td>4-0 taper RB-1 (needle comes on suture)</td>
		</tr>
		<tr>
			<td>Polydioxanone (PDS II) suture</td>
			<td>McKesson Medical-Surgical</td>
			<td>129031</td>
			<td>4-0 reverse cutting FS-2</td>
		</tr>
		<tr>
			<td>Polyglactin 910 (Vicryl) suture</td>
			<td>Butler</td>
			<td>7233-41</td>
			<td>3-0 taper RB-1</td>
		</tr>
		<tr>
			<td>Polyglactin 910 (Vicryl) suture</td>
			<td>McKesson</td>
			<td>104373</td>
			<td>4-0 reverse cutting FS-2</td>
		</tr>
		<tr>
			<td>Rabbit Chow (Alfalfa)</td>
			<td>LabDiet</td>
			<td>5321</td>
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		<tr>
			<td>Rabbit Restrainer</td>
			<td>VWR</td>
			<td>10718-000</td>
			<td></td>
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		<tr>
			<td>Rib Cutters</td>
			<td>V. Mueller</td>
			<td></td>
			<td></td>
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			<td>Scalpel</td>
			<td>Fine Science Tools</td>
			<td>10003-12</td>
			<td></td>
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		<tr>
			<td>Scalpel Blade</td>
			<td>Fine Science Tools</td>
			<td>10015-00</td>
			<td>#15 blade</td>
		</tr>
		<tr>
			<td>Silk Sutures</td>
			<td>Ethicon-Johnson &#38; Johnson</td>
			<td>A183H</td>
			<td>4-0 silk ties 18&#34;</td>
		</tr>
		<tr>
			<td>Stainless Steel Ball</td>
			<td>McMaster-Carr</td>
			<td>1598K23</td>
			<td>3-mm diameter</td>
		</tr>
		<tr>
			<td>Surgical Drapes</td>
			<td>Gepco</td>
			<td>8204S</td>
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			<td>Syringe Pump</td>
			<td>DRE Veterinary</td>
			<td></td>
			<td>Versaflow VF-300</td>
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		<tr>
			<td>Visipaque contrast media</td>
			<td>McKesson Medical-Surgical</td>
			<td>509055</td>
			<td></td>
		</tr>
		<tr>
			<td>Weitlaner Retractor</td>
			<td>Fine Science Tools</td>
			<td>17012-13</td>
			<td></td>
		</tr>
	</tbody>
</table>

preclinical model of hind limb ischemia in diabetic rabbits

Peripheral vascular disease is a widespread clinical problem that affects millions of patients worldwide. A major consequence of peripheral vascular disease is the development of ischemia. In severe cases, patients can develop critical limb ischemia in which they experience constant pain and an increased risk of limb amputation. Current therapies for peripheral ischemia include bypass surgery or percutaneous interventions such as angioplasty with stenting or atherectomy to restore blood flow. However, these treatments often fail to the continued progression of vascular disease or restenosis or are contraindicated due to the overall poor health of the patient. A promising potential approach to treat peripheral ischemia involves the induction of therapeutic neovascularization to allow the patient to develop collateral vasculature. This newly formed network alleviates peripheral ischemia by restoring perfusion to the affected area. The most frequently employed preclinical model for peripheral ischemia utilizes the creation of hind limb ischemia in healthy rabbits through femoral artery ligation. In the past, however, there has been a strong disconnect between the success of preclinical studies and the failure of clinical trials regarding treatments for peripheral ischemia. Healthy animals typically have robust vascular regeneration in response to surgically induced ischemia, in contrast to the reduced vascularity and regeneration in patients with chronic peripheral ischemia. Here, we describe an optimized animal model for peripheral ischemia in rabbits that includes hyperlipidemia and diabetes. This model has reduced collateral formation and blood pressure recovery in comparison to a model with a higher cholesterol diet. Thus, the model may provide better correlation with human patients with compromised angiogenesis from the common co-morbidities that accompany peripheral vascular disease.

Following induction of diabetes and initiation of the 0.1% cholesterol diet, the total cholesterol for the rabbits with diabetes and cholesterol diet was 123.3 &#177; 35.1 mg/dL (n = 6 male rabbits) averaged overall time points and rabbits. The BGL level for these rabbits was 248.3 &#177; 50.4 mg/dL (n = 6 male rabbits). A time course for blood chemistries and leg blood pressure ratios in a typical rabbit is shown in Figure 3 in comparison to rabbits under a higher choles...

Watch this Scientific Journal Video about Preclinical Model of Hind Limb Ischemia in Diabetic Rabbits at JoVE.com

Preclinical Model of Hind Limb Ischemia in Diabetic Rabbits

We describe a surgical procedure used to induce peripheral ischemia in rabbits with hyperlipidemia and diabetes. This surgery acts as a preclinical model for conditions experienced in peripheral artery disease in patients. Angiography is also described as a means to measure the extent of introduced ischemia and recovery of perfusion.

Peripheral vascular disease is a widespread clinical problem that affects millions of patients worldwide. A major consequence of peripheral vascular ...

A regenerative treatment for peripheral arterial disease would be extremely beneficial and this animal model may be able to better test whether experimental treatments could work in human patients. Our procedure aims to put forth an improved model. One that more accurately replicates the therapeutic resistance encountered in patients with diabetes or hyperlipidemia.This animal model can be used to test new therapies for peripheral ischemia and provides a pre-clinical model that has compromised neovascularization to ischemia. After confirming a lack of response to hind paw pinch in an anesthetized four to six month old diabetic and hyperlipidemic rabbit, use a scalpel with a number 15 blade to make a four to five centimeter long incision just lateral to the right side of the trachea and use blunt dissection to expose the right common carotid artery. Use small Weitlaner retractors to open the incision and carefully isolate the carotid artery from the jugular vein and vagus nerve.When the carotid artery has been fully separated from the nerve and jugular vein, place a 4-O silk suture at the proximal and distal ends of the exposed artery and tie off the distal end of the carotid with a surgeon's knot and four square knots. On the proximal end, use a vascular silicone tie to allow for tightening or loosening of the vessel, as needed. Next, apply approximately 0.5 milliliters of 1%lidocaine along the exposed carotid artery and nerve to promote vasodilation and reduce nerve irritation.Administer 500 international units of heparin IV.Place a four-inch wire insertion tool into the artery. Feed a 0.014 inch by 185 centimeter guidewire through the insertion tool to the aortic bifurcation at the iliac crest in the descending aorta, and advance a three French pigtail angiographic catheter over the wire. Advance the pigtail catheter to two centimeters proximal to the aortic bifurcation at the iliac crest in the descending aorta, positioning the tip of the catheter between the seventh lumbar and the first sacral vertebra.Test the location of the catheter by manually injecting two to four milliliters of contrast agent. Once correct positioning is confirmed, administer an intraarterial injection of 100 micrograms of nitroglycerin through the catheter to increase vasodilation. Then administer 0.8 to one milliliter of 1%lidocaine through the catheter to assist with vasodilation and flush the catheter with heparinized saline.Attach the tubing for the automated angiographic injector to the catheter, being sure to remove any air from the line. Use the automated angiographic injector to deliver nine milliliters of contrast medium through the catheter at three milliliters per second and perform digital subtraction angiography at six frames per second. Select the serial images and alter the image of the angiogram using minus 40%setting to minimize the appearance of bone and capture a complete picture of the vessel perfusion with contrast.To isolate the femoral artery, use a number 15 scalpel blade to make a longitudinal skin incision over the right femoral artery, making sure that the incision extends inferiorly from the inguinal ligament and ends at the area just proximal to the patella. Use blunt dissection to expose the femoral artery and open the incision with Weitlaner retractors. Apply approximately 0.5 milliliters of 1%lidocaine locally to reduce nerve irritation and promote vasodilation.Continue the blunt dissection of the tissues to free the entire length of the femoral artery along with all of its branches, including the inferior epigastric, deep femoral, lateral circumflex and superficial epigastric arteries. Dissect along the popliteal and saphenous arteries, as well as the external iliac artery, periodically hydrating the area with saline to protect against tissue damage. Carefully separate the femoral artery from the femoral vein and nerve and place two 4-O silk sutures on the femoral artery with just enough space between the sutures to allow the artery to be severed.Then use small Metzenbaum scissors to cut between the two ties of the ligated artery. Excise the femoral artery from where the artery bifurcates to form the saphenous and popliteal arteries, working proximately, tying off any side branches to excise the rest of the femoral artery to just below the epigastric artery. For repeat angiography, use a silastic sheet with a mounted three millimeter stainless steel ball.To attach the stainless steel ball into the upper part of the quadriceps muscle with 4-O silk sutures. Pull the skin over the ball when it is in place. Administer an intraarterial injection of 100 micrograms of nitroglycerin through the catheter to increase vasodilation.If needed, administer another 0.8 to one milliliter of 1%lidocaine through the catheter to assist with vasodilation and flush with heparinized saline. Use the automated angiographic injector at the same settings to inject another nine milliliters of contrast medium. Then perform another angiogram, as demonstrated.At the end of the procedure, remove the catheter from the right artery and tie off the artery using the 4-O silk suture already in place around the artery. Then close the muscle and subcuticular layers with 4-O polydioxanone or 3-O polyglactin 910 sutures on a taper needle in a continuous suture pattern and close the skin using 4-O polydioxanone or 4-O polyglactin 910 sutures on a reverse cutting needle in a buried continuous subcuticular suture pattern. In non-diabetic animals, even with higher cholesterol, there is an increased recovery of the blood pressure in the ischemic limb and vascularity in the angiograms at the final time point compared to diabetic animals.Histologically, changes in the muscle structure consistent with edema and ischemic damage are observed as a loss or disruption of the muscle fibers. Further, immunostaining for PECAM one and alpha smooth muscle actin can be used to identify the number and size of the vessels in the tissue sections. Other imaging methods, such as Doppler ultrasound, laser Doppler imaging, infrared thermography, and MRI can be added for further monitoring of the ischemic limb reperfusion.With this incorporation of therapeutic resistance, our model more accurately mimics ischemia in a diseased state, advancing the assessment of new therapies for peripheral arterial disease.

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JoVE Journal

Medicine

9.5K vistas.  University of Texas at Austin. Un tratamiento regenerativo para la enfermedad arterial periférica sería extremadamente beneficioso y este modelo animal puede ser capaz de probar mejor si los tratamientos experimentales podrían funcionar en pacientes humanos. Nuestro procedimiento tiene como objetivo presentar un modelo mejorado. Uno que replica con mayor precisión la resistencia terapéutica encontrada en pacientes con diabetes o hiperlipidemia.Este modelo animal se puede utilizar para probar nuevas terapias par...