All experimental procedures and protocols used in this investigation were approved by the Ethics Committee of Chonnam National University Medical School and Chonnam National University Hospital (CNU IACUC-H-2010-16) and conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). To prevent acute thrombosis after stenting, premedication with aspirin 20 mg per day was given for at least 4 days before the procedure.

The rabbits were randomly divided into 2 groups (Table 1): group 1 (AES, n=10) and group 2 (PES, n=10). A total of 10 rabbits were used in this study (10 rabbits, 20 iliac arteries, 10 iliac arteries in each group). An AES and a PES were implanted in the right iliac artery and left iliac artery in a randomized manner in each rabbit.

We used our developed bare metal stent (CNUH stent) as previously reported.6 The atorvastatin- and paclitaxel-loaded biodegradable PLGA [poly(lactic-co-glycolic acid)] coatings were fabricated onto the surface of the bare metal stents (CNUH stent, 3.0×18 mm, Co-Cr alloy, L605) by using an ultrasonic atomization spray method.7 The AES with 200 µg atorvastatin and PES with 177 µg paclitaxel were prepared.

Male New Zealand white rabbits (n=10) weighing approximately 3.5 kg were used. Stent implantation was performed under sterile conditions by using portable fluoroscopic imaging (BV Pulsera, Philips). Rabbits were anesthetized intravenously through the right marginal ear vein with a mixture of xylazine (2.2 mg/kg) and ketamine (22 mg/kg). Body temperature was controlled at 37℃ through the use of a heating pad and the animals received supplemental oxygen continuously through an oxygen mask. Continuous hemodynamic and surface electrocardiographic monitoring was maintained throughout the procedure.

Subcutaneous 2% lidocaine was administered at the cut-down site. An incision was made above the right carotid artery and the vessel was dissected free. A 4-Fr introducer sheath (Radifocus Introducer 2, Terumo Co, Tokyo, Japan) was inserted into the vessel, preloaded with a guide wire (Radifocus, Terumo Co, Tokyo, Japan), and advanced from the carotid artery into the distal descending aorta. The guide wire was then advanced to the proximal portion of the iliac artery. The stent delivery system was advanced to the distal portion of the iliac artery by using the guide wire and the stent was deployed to a pressure of 8 atm to achieve a stent-to-artery size ratio range of 1.3:1.8 Intravenous heparin (100 U/kg) was administered immediately after stent deployment.9 Successful deployment was verified by angiography, and the introducer sheath was removed. The right carotid artery was ligated, the incision above the carotid artery was closed in layers, and the rabbit was allowed to emerge from anesthesia. Rabbits received antibiotic prophylaxis (enrofloxacin, 5 mg/kg) for 4 days after stent implantation and aspirin in their drinking water (20 mg/day) for the duration of the protocol.9 Four weeks after stenting, the animals underwent follow-up angiography in the same views as before and were sacrificed by injection with 20 mL of potassium chloride through the left carotid artery.

The stented iliac arteries were pressure-perfusion fixed at 110 mm Hg in 10% neutral buffered formalin overnight. Arteries were step-sectioned, processed routinely for light microscopy, and stained for histological analysis.

Rabbits underwent iliac artery stent implantation as described above and were sacrificed 28 days after deployment. Histopathologic evaluation of each iliac artery was performed by an experienced cardiovascular pathologist. The specimens were embedded and sections of 50 to 100 µm thickness were obtained at about 1 mm apart and stained with hematoxylin-eosin (Fig. 1) and Carstairs' stain (Fig. 2) for histological analysis. Measurements of the histopathologic sections were performed by using a calibrated microscope, digital video imaging system, and microcomputer program (Visus 2000 Visual Image Analysis System, IMT Tech). Borders were manually traced for lumen area, area circumscribed by the internal elastic lamina, and the innermost border of the external elastic lamina (external elastic lamina area). Morphometric analysis of the neointimal area for a given vessel was calculated as the measured internal elastic lamina area minus the lumen area. The measurements were made on five cross-sections from the proximal and distal ends and the three midpoints of each stented segment. Histopathologic stenosis was calculated as 100×[1-(lesion lumen area/lesion internal elastic lamina area)].10

Arterial injury at each strut site was determined by the anatomic structures penetrated by each strut. A numeric value was assigned as previously described by Schwartz et al.10 0=no injury, 1=break in the internal elastic membrane, 2=perforation of the media, and 3=perforation of the external elastic membrane to the adventitia. The average injury score for each segment was calculated by dividing the sum of injury scores by the total number of struts at the examined section.

With regard to the inflammation score for each individual strut, the grading was as follows: 0=no inflammatory cells surrounding the strut; 1=light, noncircumferential inflammatory cells infiltrate surrounding strut; 2=localized, moderate to dense cellular aggregate surrounding the strut noncircumferentially; and 3=circumferential dense inflammatory cell infiltration of the strut. The inflammation score for each cross-section was calculated by dividing the sum of the individual inflammation scores by the total number of struts at the examined section.11 Ordinal data for fibrin were collected on each stent section by using a scale of 0 to 3 as previously reported.12

Statistical analysis was performed with the aid of commercially available software (SPSS Version 15; Chicago, IL, USA). The data were presented as mean value±SD. Unpaired Student's t-test was used for the comparison of the two stent groups. A value of p<0.05 was considered statistically significant.

The morphology of the stent surface was smooth and uniform as shown by scanning electron microscope findings. There was no destruction or delamination on the stent coating surfaces (Fig. 3). Drug release graphs showed that about 60% of the atorvastatin and 65% of the paclitaxel were released from the stent over the first 3 days. The drug release was maintained for 4 weeks in both stents (Fig. 4).

Two stents were placed in two iliac arteries per rabbit. A total of 20 stents including 10 AESs and 10 PESs were randomly placed in the left and right iliac arteries of 10 rabbits. Mortality for this study was zero. There was no significant difference in stent-to-artery ratio between the two stent groups.

There were no significant differences in the injury score (1.2±0.43 in the AES group vs. 1.3±0.45 in the PES group), lumen area (3.9±0.54 mm² in the AES group vs. 3.7±0.63 mm² in the PES group), or inflammation score (1.1±0.15 in the AES group vs. 1.1±0.10 in the PES group).

There were significant differences in the internal elastic lamina (4.6±0.46 mm² in the AES group vs. 4.2±0.55 mm² in the PES group, p<0.05), in the neointimal area (0.7±0.18 mm² in the AES group vs. 0.4±0.25 mm² in the PES group, p<0.01), in the percentage stenosis area (14.8±5.06% in the AES group vs. 10.5±6.80 mm² in the PES group, p<0.05), and in the fibrin score (0.4±0.51 in the AES group vs. 2.7±0.48 in the PES group, p<0.001) (Fig. 5).

Our study had some limitations. First, we used normal rabbit iliac arteries without atherosclerotic lesions, which is unlike the human clinical situation with preexisting atherosclerosis. Second, we examined the inflammatory reaction on the basis of H&E staining. Immunohistochemistry techniques are the standard for such studies.29

In conclusion, this study showed that although the AES did not suppress the neointima hyperplasia compared with the PES, which used a cytotoxic anticancer agent, it showed a superior arterial healing effect in a rabbit iliac artery overstretch restenosis model.