The present protocol describes a standardized surgical method for the elastase-induced AAA model through the direct application of elastase to the adventitia of infrarenal abdominal aorta in mice.
Abdominal aortic aneurysm (AAA), although primarily asymptomatic, is potentially life-threatening as the rupture of AAA usually has a devastating outcome. Currently, there are several distinct experimental models of AAA, each emphasizing a different aspect in the pathogenesis of AAA. The elastase-induced AAA model is the second most used rodent AAA model. This model involves direct infusion or application of porcine pancreatic elastase (PPE) to the infrarenal segment of the aorta. Due to technical challenges, most elastase-induced AAA model nowadays is performed with the external application rather than an intraluminal infusion of PPE. The infiltration of elastase will cause degradation of elastic lamellae in the medial layers, resulting in the loss of aortic wall integrity and subsequent dilation of the abdominal aorta. However, one disadvantage of the elastase-induced AAA model is the inevitable variation of how the surgery is performed. Specifically, the surgical technique of isolating the infrarenal segment of the aorta, the material used for aorta wrapping and PPE incubation, the enzymatic activity of PPE, and the time duration of PPE application can all be important determinants that affect the eventual AAA formation rate and aneurysm diameter. Notably, the difference in these factors from different studies on AAA can lead to reproducibility issues. This article describes a detailed surgical process of the elastase-induced AAA model through direct application of PPE to the adventitia of the infrarenal abdominal aorta in the mouse. Following this procedure, a stable AAA formation rate of around 80% in male and female mice is achievable. The consistency and reproducibility of AAA studies using an elastase-induced AAA model can be significantly enhanced by establishing a standard surgical procedure.
Abdominal aortic aneurysm (AAA) is defined as a segmental dilatation of the abdominal aorta with at least a 50% increase of vessel diameter1. AAA is potentially fatal, as the rupture can result in an extremely high mortality rate, even with intervention2,3,4. It has been reported that AAA is responsible for approximately 13,000 deaths annually in the USA, which makes it the 10th leading cause of death1,5.
The pathogenesis of AAA is not yet wholly understood6,7,8. To investigate the molecular mechanism of AAA and test potential therapeutic targets, several experimental AAA models have been established9,10. Rodent models of AAA include elastase, calcium chloride, angiotensin II and xenograft models, among which the elastase-induced AAA model is the second most used model10,11,12,13,14,15,16,17. This model involves direct infusion or application of porcine pancreatic elastase (PPE) to the infrarenal segment of the aorta. The penetration of elastase into the medial layer of the aorta will cause degradation of elastic lamellae and infiltration of inflammatory cells, leading to the loss of aortic wall integrity and subsequent dilation of abdominal aorta7,18. The elastase-induced AAA model was first reported by Anidjar et al. in 1990 using rats, in which an isolated segment of the aorta was perfused with elastase17. Later in 2012, a modified model using a periadventitial application of PPE was reported by Bhamidipati et al.19. Nowadays, most surgeries for the elastase-induced AAA model are inspired by Bhamidipati's group and are performed with the external application rather than intraluminal perfusion of PPE. Although the external application has less requirement on fine surgical skills, the incidence rate of AAA is relatively lower and size somewhat smaller than that of intraluminal perfusion11,19.
Although widely used in AAA studies, the elastase-induced AAA model possesses certain limitations. One caveat of this model is the inevitable variations of how the surgery is performed, which can lead to the issue of reproducibility. For example, the difference may exist in the surgical procedure regarding how the infrarenal segment of the aorta is isolated and which part of the segment is selected for PPE application among different laboratories. The enzymatic activity of PPE and the time duration of PPE incubation can also vary. These, however, are all essential determinants that affect the eventual AAA formation rate and aneurysm diameter.The variation of these critical determinants makes data comparison of AAA studies from different groups using this model very difficult. Therefore, a standardized surgical procedure is needed as a tool to get comparable results from various institutions.
This article describes a standardized surgical protocol for the elastase-induced AAA model through direct application of PPE to the adventitia of infrarenal abdominal aorta in mice. Details about surgical material and procedures essential for successful and robust generation of AAA in mice using this model will also be discussed.
The animal protocols were approved by the University of Michigan Institutional Animal Care and Use Committee (PRO00010092). Male and female C57BL/6J wild-type (WT) mice, ~7 weeks of age, were used for the experiments.
1. Animal preparation
2. Preparation for surgery
3. Surgery procedure
4. Post-operative care
5. Measurement of abdominal aorta aneurysm diameter
A total of twenty-three 7-week-old wild-type (WT) mice, including 12 females and 11 males, were operated following the presented protocol. The survival rate was 100% (surgical mortality excluded). Maximal abdominal aorta diameter was measured by a caliper.
AAA was defined as dilating the abdominal aorta with a 50% vessel diameter increase. Therefore, a 50% increase in the maximal abdominal aorta diameter was selected as the cut-off point for successful AAA induction. Based upon this criterion, the incidence rate of AAA on post-operative Day 14 was 91.7% for female animals, with 11 out of 12 that underwent surgery developed AAA; while the incidence rate for male animals was 72.7%, with 8 out of 11 that underwent surgery developed AAA (Figure 1A). Among the 19 animals in which AAA occurs, there is a 1.7-fold and 1.6-fold increase of maximal abdominal aorta diameter in females and males compared to non-surgery controls, respectively (Figure 1B). Representative images of the aortas from non-surgery and surgery groups on post-operative Day 14 are shown in Figure 1C.
Figure 1: Characterization of AAA in mice undergone surgery. All measurement is done on post-operative Day 14. (A) The incidence rate of AAA in mice that underwent surgery. (B) Maximal diameter of the infrarenal aorta in mice from both non-surgery (n = 4 for both female and male) and surgery groups (n = 12 for female; n = 11 for male). Data are mean ± SEM. *p < 0.05. (C) Representative images of aortas in mice from both non-surgery and surgery groups. Scale bar = 2 mm. Please click here to view a larger version of this figure.
The elastase-induced AAA model was first reported by Anidjar et al. using rats in 199017. A variety of modified versions have been introduced in the past thirty years, along with significant improvement in the surgical techniques19,20,21,22. Hundreds of institutes use elastase-induced AAA models as the second most used rodent experimental model for AAA studies12. It is only natural that different groups will perform the surgery for this model slightly differently. As a result, one caveat of this model is the inevitable variations of how the surgery is performed, which makes the comparison of data from different groups using this model very difficult. The variations include but are not limited to how infrarenal segment of the aorta is exposed, how extensively the fascial layer covering the aorta is cleaned, how long the incubation period is, as well as the material used for aorta wrapping and elastase incubation and the volume of elastase added onto the wrapping material. These, however, are all essential determinants that affect the eventual incidence rate and diameter of AAA.A standardized surgical procedure is needed to solve the issue of reproducibility caused by these variations. A standardized surgical protocol is presented here for the elastase-induced AAA model through direct application of PPE to the adventitia of the infrarenal abdominal aorta in the mouse.
Aside from the consistent induction rate of AAA, this protocol owns the advantages of low expenses and short operation time. The surgical material used in this protocol can all be easily obtained at low costs. The average operation time of this protocol is 50 min, including 30 min of PPE incubation. These advantages will allow the easier conduction of many animal studies, contributing to more substantial statistical power and reproducibility.
Exposure of the abdominal aorta is the most critical step in this protocol. It is essential to remove the connective tissue in the vessel sheath that surrounds the abdominal aorta and IVC so that elastase will have a better chance of penetrating the medial layer of the aorta. However, completely removing these connective tissues is time-consuming and risks damaging the underlying aorta and IVC. This is the step from which most surgical mortality is caused. If IVC or aorta is damaged, most animals will not survive the following 48 h even if they survive the surgery, possibly due to the continuous bleeding after surgery leading to exsanguination. For this reason, a delicate balance between exposing the aorta to the greatest extent and not damaging the big vessels needs pondering over. Leaving a small amount of connective tissue on the vessel sheath will significantly reduce perioperative mortality while not affecting AAA's occurrence. Therefore, it is not recommended to completely remove the connective tissue covering the aorta and IVC for exposure to the abdominal aorta. Still, extreme caution needs to be taken during the abdominal aorta and IVC dissection from adjacent connective tissue.
Anatomically, the abdominal aorta and IVC are closely attached via a thin fascia layer and contained together in the same vessel sheath. Some experimental models such as venous thrombosis may require separating the aorta from adjacent IVC. However, it is unnecessary to do so in the elastase-induced AAA model23. Some may prefer to rotate the vessel sheath clockwise so that the aorta will be right above IVC, and elastase can then be applied directly to the aorta. This is unnecessary as the cotton pad beneath is saturated with elastase, and the aorta will be continuously exposed to elastase regardless of its anatomic relationship to IVC. It is recommended that the backside of the aorta and IVC be adequately separated from the underlying muscles via forceps. In case the dissection is not extensive enough in this step, tension force will be created by the surrounding fascia and muscles on the stripe and cotton pad, resulting in their twist and subsequent inefficient absorbance of elastase.
One limitation of this protocol is the eventual shape of the induced AAA. It is inevitable that when being dropped onto the aorta, some elastase will flow to other segments of the aorta that are not wrapped by the cotton pad. In addition, nearby bleeding caused by dissection, even of small amount, can wash elastase away from the cotton pad to other aorta segments during the 30 min incubation period. These will lead to elastase penetrating other segments of the aorta, thus causing the breakdown of the medial layer and dilation of the abdominal aorta not limited to the segment wrapped by the cotton pad. As a result, instead of exhibiting fusiform-shaped aneurysms caused by focal dilation, some animals demonstrate general dilation of a long segment of the abdominal aorta. However, the maximal aorta diameter in these animals that have continuous dilation of the aorta still shows a more than 50% increase when compared with controls. Meanwhile, no difference in maximal aorta diameter is observed between animals exhibiting continuous dilation and focal dilation of the abdominal aorta, suggesting that the aneurysm's shape is an indicator of neither successful AAA induction nor actual AAA size in this case protocol.
Another limitation of this protocol is applying this method to larger animals. The topical elastase-induced AAA model should be easily applied to other small animals such as rats. However, elastase alone may not be enough for larger animals like pigs or primates to induce a consistent AAA phenotype. For example, Marinov et al. reported that elastase perfusion alone in swine could cause some histologic changes, including elastin disruption, but could not induce consistent aneurysm24. Shannon A et al. reported a porcine model of infrarenal AAA induced by elastase. However, it requires a combination of balloon angioplasty, perfusion, topical elastase, and a diet containing BAPN (a lysyl oxidase inhibitor preventing collagen crosslinking) given the considerable durability of aorta25. Therefore, it seems the application of a standardized elastase-induced AAA protocol in larger animals is still quite challenging due to the difference in size and durability of the aorta between mice and larger animals, the technical challenges, and the potentially high cost.
In summary, despite some limitations, AAA can be induced consistently in both male and female mice in this standardized protocol. The consistency and reproducibility of AAA studies using the elastase-induced AAA model are expected to be significantly enhanced by establishing this standard surgical procedure.
The authors have nothing to disclose.
We thank the Unit for Laboratory Animal Medicine of the University of Michigan for their help with animal feeding and breeding. This study is supported by NIH RO1 HL138139, NIH RO1 HL153710 to J. Zhang, NIH RO1 HL109946, RO1 HL134569 to Y.E. Chen, and the American Heart Association grant 20POST35110064 to G. Zhao.
6-0 non-absorbable monofilament suture | Pro Advantage | P420697 | |
Carprofen | Zoetis Inc. | NDC: 54771-8507 | |
Chow Diet | LabDiet | 3005659-220 | PicoLab 5L0D |
Cotton Applicator | Dynarex | 4303 | |
Cotton Pad | Rael | UPC: 810027130969 | |
GraphPad Prism 8 | GraphPad Software Inc. | Version 8.4.3 | |
Grarfe Forceps | Fine Science Tools | 11051-10 | |
Halsted Mosquito Hemostats | Fine Science Tools | 13009-12 | |
Ketamine | Par Pharmaceutical | NDC: 42023-0115-10 | |
Nitrile gloves | Fisherbrand | 19-130-1597 | |
Penicillin-Streptomycin | Thermo Fisher | 15140122 | |
Porcine pancreatic elastase | Sigma-Aldrich | E1250-100MG | |
Scissors | Fine Science Tools | 14068-12 | |
Sterile 0.9% saline solution | Baxter | 2B1324X | |
Xylazine | Akorn | NDC: 59399-110-20 |