Experimental animals
A total of 16 male Sprague–Dawley (SD) rats that were six weeks of age and weighed 180-200 g, were included. Rats were randomly divided into 4 groups (4 rats in each group): group I (control diet, saline infused, control water), group II (control diet, porcine pancreatic elastase (PPE) infused, control water), group III (hypercholesterol diet, PPE infused, control water), and group IV (hypercholesterol diet, PPE infused, water mixed with statin) [10, 11]. The control diet was both low fat and low cholesterol (D12337) (Research Diets, New Brunswick, NJ, USA), while the hypercholesterol diet was a rat chow designed to be high in cholesterol (Paigen’s atherogenic rodent diet, D12336) (Research Diets, New Brunswick, NJ, USA) [10]. Atorvastatin (Lipitor, Pfizer, NY, USA) (0.2 μg/g body weight/day) was administered by standard oral gavage in group IV for 5 weeks [1].
Surgical Procedures
During the third week, rats were anesthetized using 2% inhaled isoflurane, and surgery was carried out by a vascular surgeon as follows [11,12,13]. Periaortic dissection was performed from renal vein level to bifurcation of aorta (Fig. 1A). The initial aortic diameter (AD1) was measured with a micrometer caliper in all groups. Aortic branches such as lumbar arteries were ligated with suture (polypropylene 8-0) (Prolene, J&J, NJ, USA) (Fig. 1B). Three silk sutures (4-0, Ethicon, J&J, NJ, USA) were separately prepared for three ties at the renal vein level, incision point, and bifurcation of aorta (Fig. 1C). At first, two ties at renal vein level and bifurcation of aorta were performed to minimize procedural bleeding. The initial ties were followed with a small incision placed above a tie at the bifurcation site of the aorta, followed by insertion of the angiocatheter (26G, Polypen, Poly Medicure, Brussels, Belgium). The third tie of aorta was performed to fix the angiocatheter in place and minimize bleeding between the angiocatheter and the incision of aorta (Fig. 1D). The isolated region of the aorta was then injected with saline in group I or type I PPE (30 U/ml) (5.9 U/mg; Sigma Aldrich, St. Louis, MO, USA) in group II-IV [11, 12]. Using 10 ml of saline in group I or a 10 ml saline solution mixed with 3 ml PPE in group II-IV, approximately 1 ml of solution was loaded in the aorta for 5 min with an infusion pump operated at 2 atmospheric pressure [9, 11, 12]. During this period, expanded aortic diameter (AD2) was measured with a micrometer caliper in all groups. All ties were removed along with the angiocatheter after successful completion of the perfusion, followed by closure of the incision using a suture (polypropylene 8-0) (Prolene, J&J, NJ, USA) [11, 12]. Recovery of blood flow in the aorta and common iliac artery (Fig. 1E) were performed and ischemic time and procedural time were checked. Ischemic time is equal to aorta clamping time in the current study.
At the 5th week, aneurysmal change was inspected (Fig. 1F) and the final aortic diameter (AD3) was measured using a micrometer caliper after aortic dissection via laparotomy under anesthesia using 2% inhaled isoflurane. A 1 cm-sized infrarenal segment of aorta and 1 ml blood was obtained from each rat. Rats were euthanized with carbon dioxide according to the guidelines of our institute [10].
Laboratory and histologic analysis
Body weights of all rats were measured at the first and fifth week. Total cholesterol (TC), high density lipoprotein (HDL), low density lipoprotein (LDL), and triglyceride (TG) levels were evaluated in blood samples from each rat. Hematoxylin and eosin (H&E) staining, and immunohistochemistry (IH) with alpha smooth muscle actin (α-SMA) antibody were carried out on embedded aortic tissue sections cut following fixation in 10% formalin solution [10]. The degree of cellularity was assessed at a magnification of 400 (× 400) using a magnified-high power field (HPF) microscope. The number of inflammatory cells in media and adventitia was measured twice by examiners blind to group assignments [10].
Immunohistochemistry (IH) with alpha smooth muscle actin (α-SMA) antibody
For detection of α-SMA in tissues, tissue sections were incubated with 0.5% TritonX-100 (Sigma Aldrich, St. Louis, MO, USA) in 0.01 M phosphate-buffered saline (PBS) and blocked with normal donkey serum [10]. Subsequently, the tissue sections were incubated overnight at 4 °C with primary antibodies against α-SMA [10]. Sections were rinsed in PBS and incubated in peroxidase-conjugated donkey anti-mouse or rabbit antimouse IgG (Amersham Pharmacia Biotech, Piscataway, NJ, USA) [10]. Following a rinse in Tris-buffered saline (TBS) (Sigma Aldrich, St. Louis, MO, USA), the tissue sections were incubated with a mixture of 0.05% 3,3-diaminobenzidine until a brown color was visible, washed with TBS, counterstained with hematoxylin, and examined by light microscopy [10].
Statistical analysis
Statistical analysis included Student t-test, Fisher's exact test, Wilcoxon test, and ANOVA using SPSS 20.0 (IBM Inc., Armonk, NY, USA). A P value of less than 0.05 was considered statistically significant. Data are presented as mean ± standard deviation.
This research was approved by the Institutional Animal Care and Use Committee (IACUC) of our institute (CIMH-2019-020).