Name: preclinical model of hind limb ischemia in diabetic rabbits
Uploaded: 2019-06-02T13:00:00
Description: Watch this Scientific Journal Video about Preclinical Model of Hind Limb Ischemia in Diabetic Rabbits at JoVE.com

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
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	<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.

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</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 ...

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