All animal protocols were approved by the Chonnam National University Animal Care and Use Committee (CNU IACUC-H-2009-18). Male Sprague-Dawley rats were purchased from Samtako (Korea). All rats (b.w. 400 ± 20 g) were fed a normal pelleted diet and were given water ad libitum. Starting 1 week before stent implantation and continuing throughout the study period, the rats' drinking water contained aspirin (3.25 mg/kg; Bayer Vital, Germany) and clopidogrel (0.75 mg/kg, Plavix; Sanofi-Aventis, USA).

In the baseline study, a total of 14 rats underwent an aortogram and surgery. We measured the aorta size of 10 rats by angiogram before stent implantation. To select the optimal stent size (diameter and length), balloon pressure, and stent-to-artery ratio, the aorta size of the rats that underwent stent implantation was measured by angiogram. A balloon pressure of 9 atm resulted in a stent-to-artery ratio into 1:1.2. A BMS (n = 6, Liberté 3.5 × 20 mm; Boston Scientific, USA) or a PES (n = 6, Taxus Liberté 3.5 × 20 mm; Boston Scientific, USA) was deployed at 9 atm in the rats.

For stent implantation, the rats were anesthetized with an intraperitoneal injection of ketamine (50 mg/kg; Bayer Animal Health, Korea) and xylazine (6.7 mg/kg; Bayer Animal Health, Korea), and the left common carotid artery was surgically exposed. The proximal portion of the left common carotid artery was ligated with 5-0 silk thread. The distal portion was then occluded by a vessel clamp to block blood flow and a carotid cut down was performed at the mid portion. Under fluoroscopic guidance [7,18], the stent catheter was advanced to the thoracic aorta via the left common carotid arteriotomy. A 0.36 mm percutaneous transluminal coronary angioplasty (PTCA) guide wire was advanced into the thoracic aorta. A BMS or PES with a balloon-to-artery ratio ranging from 1:1.2 to 1:1.3 was placed at the desired location in the thoracic aorta and inflated to 9 atm of balloon pressure. After stent deployment, the animals were allowed to recover in their normal housing at the animal care facility. The animals were monitored daily until the subsequent experimental procedures and euthanasia 2 or 4 weeks after stent placement.

Aortography was performed 2 or 4 weeks after stent deployment (Fig. 1A) using a contrast agent (Visipaque; Amersham Health, Ireland). An angiography catheter (18 G radiology needles; BD, USA) was introduced through the right common carotid artery and advanced to the thoracic aorta to deliver the contrast agent. Aorta size was calculated by quantitative angiography before stent implantation. Stent size and balloon pressure were also determined. The stent-to-artery ratio was calculated by dividing the post-implant aorta diameter by the pre-implant aorta diameter.

At 2 and 4 weeks after stent implantation, the rats were anesthetized with an intraperitoneal injection of ketamine and xylazine. After the final aortogram, the aorta was flushed gently with a 0.9% saline solution and perfusion fixation was performed with 2.5% glutaraldehyde. The stented aorta was harvested with at least 1 cm of the proximal and distal margin. After an additional 24 h of immersion fixation with 2.5% glutaraldehyde, the stented aorta was cut into two segments.

One segment of the stented aorta was used for scanning electron microscopy (SEM) analysis. SEM analysis was conducted by following standard procedures. In brief, the stented aorta was fixed with 2.5% glutaraldehyde in 0.1 M cacodylate buffer (Sigma Aldrich, USA) for 2 h and then OsO₄ for 90 min, followed by dehydration through a series of alcohols. The samples then underwent critical point drying before being coated with evaporated gold and observed under an S-3500N scanning electron microscope (Hitachi, Japan). The re-endothelialization ratio was calculated by using NIS-Elements (version 3.00 SP7; Nikon, Japan).

The other segment of the stented aorta was placed in 10% formalin for an additional 24 h. Cross-sections were prepared from the segment by embedding the tissue in acrylic plastic and cutting sections 75 µm-thick. All sections were stained with hematoxylin and eosin. Histomorphometric analysis was carried out using Nis-Elements. The neointimal area was calculated as the media area minus the inner luminal area (neointimal area = media area - inner luminal area). These areas were determined in each section, averaged, and expressed as the absolute area in mm². Additionally, the uncovered strut ratio was determined by microscopic observation and calculated as exposed strut number / total strut number. The focal thrombus ratio was calculated as thrombus-containing strut number / total strut number as found by microscopic observation. These were averaged over all stained sections. At SEM analysis, the re-endothelization percentage (%) was calculated as the strut area minus the uncovered area divided by the strut area (re-endothelization percentage (%) = strut area - uncovered area / strut area) (Fig. 4Ab).

Measurements for each stented aorta (total sections from the mid to distal portion) were averaged to produce a mean value per stent. Tests for statistical significance were two-tailed, and significance was established by a p-value < 0.05 (v 16.0; SPSS, USA). Unless otherwise noted, data are represented as the mean ± SD.

The total procedure time for stent implantation in the rat aorta was approximately 15 to 20 min. Four rats died during surgery due to bleeding from the carotid artery at the site of incision. All stented aorta (n = 12) in the surviving rats were patent. The operation procedure is illustrated in Fig. 1A. The left common carotid artery was exposed and PTCA guide wire was inserted into the carotid artery. The stent was then inserted into the thoracic aorta via the guide wire (Figs. 1B and C). At 2 or 4 weeks after stent implantation, angiography was performed with an 18 G IV catheter in the right carotid artery (Fig. 1D). Quantitative angiograms at each time point (2 or 4 weeks after stenting) showed that the stents were implanted with similar stent-to-artery ratios.

The neointimal area was larger in the BMS group (0.262 ± 0.010 mm²) than in the PES group (0.170 ± 0.014 mm²) at 2 weeks following stent placement (p < 0.05) (Figs. 2A and 3A). However, no significant difference in the neointimal area was observed between the two groups [BMS group: 0.567 ± 0.055 mm² vs. PES group: 0.618 ± 0.089 mm² (p > 0.05)] at 4 weeks (Figs. 2A and 3C). The uncovered strut ratio was 38.3 ± 16.5% in the BMS group and 84.2 ± 1.1% in the PES group at 2 weeks, and 8.3 ± 4.0% in the BMS group and 45.0 ± 7.1% in the PES group at 4 weeks (Fig. 2B). The focal thrombus ratio was 0.0 ± 0.0% in the BMS group and 29.4 ± 8.9% in the PES group at 2 weeks and 5.0 ± 7.1% in the BMS group and 51.3 ± 8.3% in the PES group at 4 weeks. In addition, thrombi were detected only in the neointimal area (Figs. 2C and 3D).

At SEM analysis, the portion of the stent struts that remained uncovered was less in the BMS group than in the PES group at 2 weeks after stent implantation. Stent detachment was observed in the PES group (Fig. 4B). As seen in Figs. 4C and D, the portion of the stent struts that was covered increased in both groups at 4 weeks. The BMS struts were mostly covered (93 ± 4%), but the PES struts were not sufficiently covered (53 ± 18%) (p < 0.05). The strut was covered by endothelial cells in the BMS group (Fig. 4E, green arrows). In the PES group, non-endothelial cells such as inflammatory cells and platelets were detected on the stent surface (Fig. 4E, yellow arrows).