Murine Models of Atherosclerosis and Peripheral Vascular Disease

C. Keith Ozaki, MD and Scott A. Berceli, MD, PhD

Investigators interested in exploring the use of animals for research should familiarize themselves with National and appropriate local standards. Sites useful for proper conduct of procedures on mice include the NIH Guidelines for Survival Rodent Surgery and several vendor sites.

Why Mice?

• Advanced genetics, and array of animals available from vendors.
• Established models of human disease, such as immunodeficiencies (e.g. nude, scid) and diabetes.
• Less sentient than other more established animal models
• Relatively easy to handle, low housing costs
• Low doses/volumes required for test solutions (e.g. antibodies, gene therapy vehicles)
• Technological advances allow physiologic monitoring.
• Wide availability of reagents for use with murine tissue (i.e. - CALTAG Laboratories, Dako, SEROTEC, Vector Laboratories, and Zymed Laboratories.)

Disadvantages of Murine Models

• Performance of many technical procedures (e.g. bypasses, balloon angioplasties, arteriovenous fistulas) can be difficult and hard to reproduce due to the small size of the murine vasculature
• Antibodies are often of murine origin
• Models are new, and lack a longstanding track record
• Available tissue quantities for assays are small
• As with all non-human research, applicability to human situation may be limited

Genetic Models

In the early 1990's the development (by homologous recombination in embryonic stem cells) of apolipoprotein (apo) E-deficient mice revolutionized the use of murine models in the study of cardiovascular disease (Cell; Science). These animals spontaneously developed severe hypercholesterolemia and vascular lesions similar in appearance and distribution to those observed in humans. Labs have been quick to target specific genes of interest in Apo E-deficient mice to elucidate their role in the genesis of atherosclerosis. Additional mouse models of atherosclerosis have subsequently been developed through other perturbations in lipoprotein metabolism. The current status of this and related approaches has recently been reviewed (Curr Opinions Lipidology; J Path).

Carotid Ligation

Simple carotid ligation was described as a reproducible model of neointimal hyperplasia in 1997. Four weeks after ligation, luminal area is reduced by 80% through a combination of decreased vessel diameterand neointima formation. Recent research has further dissected the mechanisms of the formation of this neointima. Stasis initiates platelet activation, leading to microthrombosis and platelet-leukocyte conjugate formation, triggering inflammation and tissue-factor accumulation on the carotid artery endothelium. Delayed coagulation then results in formation of a fibrin matrix, which serves as a scaffold for smooth muscle cells. This model holds several key advantages, including technical simplicity and presence of an intact vascular endothelium. It has been used to explore the role of selectins and cytokines in the development of neointimal hyperplasia. One word of caution - we (as well as the group that originally described this model) have observed significant variation between mouse strains with regards to the intimal response in this model.

Arterialized Vein Patch

Arterialization of vein grafts can result in pathologic occlusive lesions. Building on a method employed in larger animals, authors recently used a patch cut from the external jugular vein of a mouse to repair a surgically created defect in the carotid artery. This arterialized vein patch develops accelerated neointimal hyperplasia, and may serve as a useful tool for the dissection of the mechanisms of vein graft failure.

Vein Bypass Grafts and Arterial Allografts

Vein arterial bypass grafts and arterial allografts grafts in mice offer a powerful tool for directly studying specific genes in vein graft failure and transplant atherosclerosis. We have found that high quality, well maintained instruments are crucial for successful completions of these models. Most papers report use of a vein cuff technique for anastomosis constructions, using either the vena cava or jugular vein from a donor animal as conduit (JCI; ATVB; Circ Res1; Circ; Circ Res2). Arterial allografts, in combination with the cuff anastomosis, have also been used to investigate the mechanisms underlying transplant arteritis.

Wire Injury

An early description of a murine carotid artery mechanical injury model came from the University of Washington. However, technical difficulty in producing a standardized injury with reliable neointimal lesion has limited its widespread applicability. Dr. Reidy's group continues to work with similar models, including carotid endothelial denudation using a plastic filament. The femoral artery has recently been described as another site amenable to wire injury.(J Mol Cell Card; J Vasc Surg) If the wire is introduced through a branch of the femoral artery then blood flow across the site of injury is preserved. In addition to endothelial denudation, some of these methods may distend the artery, similar to a full wall thickness angioplasty injury.

Closed Intimectomy

In an effort to denude the vascular endothelium without the hazards of opening the tiny murine artery, the author experimented with a "closed intimectomy" device (Circulation 1996; 94(8):403). Although the exposed sub-intimal surface re-paved with a confluent endothelial cell monolayer within two weeks, only 13% of specimens developed appreciable neointimal hyperplasia, thus limiting its usefulness.

Air Desiccation and Distension

As has been performed in larger animals, arterial lumen air desiccation and distension in mouse arteries results in intimal injury and neointimal lesions.

Chemical Injuries

Oxidizing agents such as ferric chloride can be applied adventitially to murine arteries to induce platelet-rich thrombi and full thickness wall necrosis. This resorbing clot serves as the scaffolding for myofibroblast migration, leading to neointimal lesions that contain smooth muscle a-actin positive cells (Surg Forum, 1996; 47:336-339). However, the mechanisms of this process may have little resemblance to most clinically significant chronic arterial occlusive pathologies.

Electrical Injury

Carmeliet et al introduced a perivascular electrical injury model of neointimal hyperplasia in 1997. This severe injury results in nearly full thickness arterial wall injury, with wound healing proceeding through cellular ingrowth from adjacent artery. The potential power of such a murine model was then demonstrated by the same group through investigation of mice with a disrupted Plasminogen gene.

Photochemical Injury

A Japanese group with extensive photochemical experience used the photochemical activation of rose Bengal by incident light at 540 nm to induced neointimal lesions. They utilized this approach to study the effects of the phosphodiesterase inhibitor milrinone on intimal hyperplasia.(Science)

Perivascular Biomaterial Cuff

Loose placement of a non-flow obstructing polyethylene cuff around the murine femoral artery results in reproducible intimal growth. An advantage of this approach is that the endothelium remains undisturbed.