Polyurethane catheter insertion into the aortic lumen and suture ligation of the aorta induce chronic hypoxia due to hypoperfusion of the adventitial vasa vasorum. This article describes a novel animal model of abdominal aortic aneurysm (AAA) with characteristics similar to those of AAA in humans.
The adventitial vasa vasorum (VV) provides oxygen and nourishment to the aortic wall. Hypoxia in the aortic wall can cause enlarged abdominal aortic aneurysms (AAAs). This article introduces and describes a standard protocol used to induce AAAs through adventitial VV hypoperfusion created with a combination of polyurethane catheter insertion into the aortic lumen and suture ligation of the infrarenal abdominal aorta.
The protocol involves the use of male rats weighing 300-400 g, which are provided food and water ad libitum. After laparotomy with a ventral midline abdominal incision, exfoliation of the aorta is performed, which blocks blood flow from the perivascular tissue. Aortotomy involving a small incision adjacent to the renal artery branches is performed, and a polyurethane catheter is inserted using an 18-gauge indwelling needle. After repairing the incision, tight ligation of the aorta over the catheter blocks VV blood flow from the proximal direction through the aortic wall without disturbing the aortic blood flow. This technique can induce an AAA with progressive aortic dilatation.
The greatest benefit of this model is that VV hypoperfusion causes tissue hypoxia and the development of an infrarenal AAA, which has morphological and pathological characteristics similar to those of a human AAA.
The abdominal aorta is composed of the following three layers: the inner vascular wall (intima), medial layer (media), and outer vascular wall (adventitia), and of these, the adventitia has a unique blood supply system known as the vasa vasorum (VV). Aortic tissue is supplied with oxygen through adventitial VV perfusion and simple oxygen diffusion from the aortic blood flow1. However, geographically, the abdominal aorta has the least distribution of VVs compared to that in other parts of the aorta.2
A previous study reported on tissue hypoxia in human abdominal aortic aneurysm (AAA) walls with thick intraluminal thrombus (ILT)3. Additionally, it has been shown that an adventitial VV in aneurysmal walls is occluded with arteriosclerotic changes at a significantly higher rate, which is associated with tissue hypoxia in the AAA walls4. Based on these findings, a novel rodent model of AAA was created by inducing adventitial VV hypoperfusion5. In this model, VV hypoperfusion caused tissue hypoxia and the development of an infrarenal AAA, which had morphological and pathological characteristics similar to those of a human AAA6. Prime examples were the presence of ILTs and the accumulation of hyperplastic adipocytes6, and the potential to cause rupture7,8. These findings have been rarely observed in previous rodent models. Therefore, this model may greatly contribute to a deeper understanding of the mechanism responsible for AAA development and rupture. We introduce and describe a standard protocol used to induce AAAs through adventitial VV hypoperfusion, and we explain how to induce hypoxia in the aortic wall using surgical techniques.
Animal care and experiments were performed in accordance with the guidelines of the Hamamatsu University School of Medicine Animal Care Committee at the Center for Animal Care.
1. Surgical Procedure for Creating the Model
NOTE: Place the surgical instruments into a bead sterilizer for 10 s preoperatively. Use sterile gloves intraoperatively.
2. Harvesting, Fixing, and Elastica-van Gieson (EVG) Staining
The described operative techniques create a novel animal model of a chronic aortic hypoxia-induced aneurysm by using a combination of polyurethane catheter insertion and suture ligation of the infrarenal abdominal aorta in rats. The rats described in the Protocol section were euthanized 28 days after the procedure. The aortas were harvested and imaged to visualize aneurysm formation. Figure 2 shows development of the fusiform AAA. The upper and lower ends of the aorta in ex vivo have a normal diameter without dilatation. Aortic diameters were measured using transabdominal ultrasonography (Figure 3). The diameter generally reaches its maximum size at about 14 days after the procedure; thereafter, it remains unchanged or slightly increases. Figure 4 shows the histopathological image of the aneurysm at its maximum diameter after EVG staining. The image of the tissue on day 28 (aneurysm) showed prominent degradation of elastic fibers compared to that on day 0.
Figure 1: Surgical procedures to induce an abdominal aortic aneurysm (AAA).
(A) The infrarenal aorta is exfoliated from the surrounding tissue. (B) A polyurethane catheter cut 10 mm long is inserted through a small incision in the aorta. (C) The incision is repaired with an 8-0 monofilament suture and blood flow is restored. (D) The aorta is ligated with a 5-0 silk suture over the inserted catheter. Scale bar = 5 mm. Please click here to view a larger version of this figure.
Figure 2: Postoperative representative results.
Macroscopic view on postoperative day 28 showing development of fusiform abdominal aortic aneurysms. The elevated margin of the retroperitoneum corresponds to the outer edge of the aneurysm (broken lines; left). The upper and lower ends of the aorta in ex vivo are normal (right). Scale bar = 3 mm. Please click here to view a larger version of this figure.
Figure 3: Maximum aortic diameters measured using transabdominal ultrasonography.
The aortic diameter steadily increased in this rat model. Aortic diameters are presented as a mean ± standard deviation (n = 12). Please click here to view a larger version of this figure.
Figure 4: Representative images of aneurysmal tissue with Elastica van Gieson staining.
Histological evaluation with EVG staining showing the degenerative elastic lamina in the media and formation of an intraluminal thrombus 28 days after the procedure (right). Elastic fiber fragmentation in the aortic media and sparse collagen fiber in the aortic adventitia are observed on day 28. Day 0 is before the procedure (left). Scale bar = 500 µm. Please click here to view a larger version of this figure.
Under physiological conditions, the inner layers of the aortic wall are nourished by diffusion from the luminal blood flow, whereas the outer and middle layers are nourished by the VV, which penetrate from the adventitia into the medial VV1. VV blood flow into the abdominal aortic wall can originate from the following three directions/areas: (1) the proximal direction through the aortic wall, (2) distal direction through the aortic wall, and (3) perivascular tissues10. Previously, our histological analysis of human tissues identified significant stenosis or occlusion of the VV in the AAA wall, suggesting that VV blood flow into the abdominal aortic wall can be reduced4. It is an extremely important point in this protocol that an infrarenal AAA was caused by a combination of polyurethane catheter insertion and suture ligation of the infrarenal abdominal aorta. To carefully exfoliate the tissue layer, surgeons must smoothly insert a polyurethane catheter into the aorta and firmly ligate the aorta to cause chronic hypoxia due to hypoperfusion of the adventitial VV and aneurysm formation. Using these techniques, blood flow in the aortic wall is consequently decreased, and a local hypoxic environment is induced. The blood flow reduction and hypoxia-induced aneurysm formation indicates that VV blood flow into the abdominal aortic wall plays a role in the pathogenesis of AAA formation.
Specifically, an aortic aneurysm model must satisfy the following conditions: a 1.5-fold increase in the vascular diameter compared to baseline, degeneration of the tunica media, and inflammation of the aortic wall. The most popular animal models have been constructed by inducing inflammatory responses using substances, such as CaCl211, elastase12, and angiotensin II13. These models can have a high reproducibility and obviously cause pathological change, and they have been commonly used in research studies. In our model, we assessed the aortic diameter using ultrasonography every 7 days from before the procedure was performed until day 28 after the procedure (Figure 3). Results showed that the aortic diameter moderately increased over the 28 days, indicating that this change in the diameter is similar to that in previous rodent models. Gross observation of the vascular form indicated a smooth fusiform shape (Figure 2). On day 28, we sacrificed the rats and performed histopathological analysis of the aortic tissue that was recovered. Tearing and disappearance of the elastic and collagen fibers of the tunica media and adventitia were observed (Figure 4). Moreover, inflammatory cells, such as macrophages, were present from the tunica adventitia to the tunica media.
Currently, the treatment options available for AAAs are limited to surgical repair and endovascular stent grafting, with mortality rates of 30 – 50% in patients with AAA rupture14. However, no drug has been approved for clinical use to treat AAAs. There is debate that there are discrepancies in the pathological findings between humans and established animal models used in AAA research. Similarities in the pathogenesis between human AAA and animal AAA models are essential for the development of pharmacological treatments. Regarding the effectiveness of rodent models, our rat model is morphologically similar to humans in terms of intraluminal thrombus5 and adipogenesis8. Furthermore, about 20% of the rats in this study had AAA rupture and died within 28 days after the procedure. Although aortic aneurysm rupture is the most critical event for this disease, rupture is uncommon with established experimental AAA models, and the mechanism has not yet been elucidated. Therefore, this model is useful for understanding the mechanism of dilation of the aortic diameter and rupture of the aneurysm.
The creation of this model is required for some surgical procedures. Therefore, researchers must practice creating this model, which is a limitation of this model. In the future, we would like to create a rodent model in which we can decrease blood flow by gradually thickening the VV walls, resulting in spontaneous aortic aneurysm.
The authors have nothing to disclose.
This work was supported by Grants-in-Aid for Scientific Research (B) (20291958) to N.U.; Grants-in-Aid for Young Scientists (A) (25713024) to N.Z.
rat | Japan SLC.Inc | Slc:SD rat | Sprague–Dawley ratTM |
povidone-iodine solution | Libatape Pharmaceutical Co., Ltd. | 4987335 111457 | |
5-0 silk string | Akiyama Medical MFG. CO.,LTD | JIS No.1 | |
vascular clips | Natsume Seisakusho Co., Ltd. | C-42-S-2 | |
polyurethane catheter (24-gauge indwelling needle) | MEDIKIT | 24G | Supercath Z4VTM, 24-gauge indwelling needle |
polyurethane catheter (18-gauge indwelling needle) | MEDIKIT | 18G | Supercath Z3VTM, 18-gauge indwelling needle |
8-0 monofilament string | Ethicon Suture | c-42-S-2 | PROLENE Polypropylene Suture, Repair the incision with the suture |