All animal experiments were performed in accordance with a protocol approved by the Institutional Animal Care and Use Committee of Wannan Medical College.
Description of the animal model
New Zealand White rabbits of 2.5–3.0 kg (n = 60) were randomly divided into two large groups (n = 30 per group). Every large group was divided into five sub groups (n = 6 per group). The specimens from the first large group divided into subgroups A, B, C, D and E were heparinized. The specimens from the second large group were subdivided into groups a, b, c, d and e without heparin administration. Specimens from groups A, B, C, D and E as well as groups a, b, c, d and e were respectively incubated with porcine pancreatic elastase solution at the concentration of 0, 0.5, 1, 2.5 and 5 U/μL.
All rabbits were anesthetized through intravenous injection of 30 mg/kg sodium pentobarbital. The surgical exposures and creations of the fusiform aneurysms were performed strictly under sterile conditions.
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Step 1: A segment of the right common carotid artery (RCCA), approximately 2.5 cm in length, was carefully exposed and isolated as previously described [19]. A piece of sterile neurological sponges (7 × 8 mm) and a slice of latex cuff (1.2 × 2 cm) were used to isolate the exposed carotid artery (Fig. 1a, d).
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Step 2: The proximal and distal portions of the latex cuff were temporarily ligatured around the carotid artery with 4.0 silk sutures. After that, different concentrations (0.5, 1, 2.5 or 5 U/μL) of porcine pancreatic elastase (≥30 units/mg, pH 8.1–8.9, PI 9.5, Shanghai Kayon Biological Technology Co., Ltd., Shanghai, China) solution were perfused into the latex cuff through 24G venous catheter until the latex cuff was filled entirely to coat the vessel and maintained for 20 min (Fig. 1b, e).
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Step 3: The elastase solution in the latex cuff was absorbed with sterile cotton gauze 20 min later. The temporary ligatures, neurological sponges and the latex cuff were removed. Then, the arterial segment and incision was irrigated with physiological saline to clear any residual elastase. Finally, the fusiform vessel segment was examined and the surgical site and incision was closed (Fig. 1c, f).
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Specimens from the control groups also underwent the same above mentioned steps. The unique difference is that the carotid artery segments were incubted with physiological saline rather than elastase solution.
All animals in the first large group were administered with heparin (2000 U/Kg) intravenously after the RCCA were exposed and prior to the installation of the elastase. The same dose was applied once per day for the next 3 days. All animals in the second large group were not administered with heparin. All animal specimens in the study were given antibiotic injections to prevent infections post surgical exposure.
Intravenous digital subtraction angiography
All rabbits except for the samples lost to attrition underwent an intravenous digital subtraction angiography (IVDSA) through auricular veins 3 weeks post-surgery using 3-5 ml angiographic contrast injections through a 24 gauge venous catheter under anesthesia. The size of aneurysm in our experiment was defined through the inner diameter of the dilated segment (that is the greatest dimension of the transverse minor axis of the fusiform segment). The images and measurements were evaluated by an independent observer who was blinded to other groups.
Histological analysis
Immediately after IVDSA, all specims were sacrificed through an overdose of sodium pentobarbital (120/mg/kg). The specimens from these animals were then acquired after in vivo fixation with 10% phosphate-buffered formaldehyde solution perfused at 100 mmHg maintained for 20 min. From the proximal to the distal end, each specimen was cut into continuous slices of 5 μm. Next, these slices were stained with hematoxylin and eosin (H&E) for general appearance and elastic van-Gieson (EVG) dye for elastin. Images of the sections were analyzed by using Image-pro plus 6.0software. The wall thickness was measured as the average thickness of 10 points along the cross-section area in the H&E stained sections.
Immunohistochemistry analysis
Tissue sections in group E were de-paraffinized in xylene, rehydrated through graded alcohol washes, and incubated with 1% H2O2 in methanol maintained for 10 min to block endogenous peroxidase activity. Non-specific binding was blocked with bovine serum (A8020, Solarbio, Beijing, China) for 20 min at room temperature, and then CD31 (ready-to-use, ab28364, Abcam) antibodies were incubated overnight in a humid chamber at 4 °C overnight. After a wash in phosphor buffered saline (PBS), sections were incubated with biotinylated anti-mouse second antibody (Boster, Wuhan, China) for 20 min followed by the SP method according to the manufacture’s protocol. Diaminobenzidine tetrahydrochloride was used to visualize the sections and counterstaining with hematoxylin was performed, after that the sample was coverslipped.
Immunofluorescent analysis of smooth muscle cells
After blocking endogenous peroxidases, sections in group E were incubated at 4 °C overnight with mouse monoclonal anti-alpha smooth muscle actin (1:150 diluted in PBS,GB11001), followed by incubation with Cy3-conjugated goat anti-mouse IgG (Google Biological Technology Co., Ltd., Wuhan, China) to identify smooth muscle cells (SMCs).
Statistical analysis
All data were expressed as mean ± standard deviation. Significant differences between different groups were determined by 2-way ANOVA-test (GraphPad Prism 5.01). A p-value of <0.05 was considered significant.