The animal study was approved by the Ethics Committee of Chonnam National University Medical School and Chonnam National University Hospital (CNU IACUC-H-2010-1), 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). Study animals were female swine weighing 25-35 kg.

The dual-coated stent was prepared by grafting abciximab and ALA to a bare metal stent (3.0×18 mm, MAC®stent, AMG, Munich, Germany) which had been coated with a polymer layer by plasma polymerization of 1,2-diaminocyclohexane (DACH) using the method of Song et al.17)(Fig. 1).

To prevent acute thrombosis after stenting, premedication with aspirin (100 mg/day) and clopidogrel (75 day/mg) was given for 7 days before the procedure.

On the procedure day, pigs were anesthetized with ketamine {20 mg/kg intramuscularly (IM)} and xylazine (2 mg/kg IM). They received IM ketamine every 30 minutes and supplemental oxygen continuously through an oxygen mask. Subcutaneous 2% lidocaine was administered at the cut-down site, left carotid artery was surgically exposed, and a 7 French sheath was inserted.

Continuous hemodynamic monitoring and surface electrocardiographic monitoring were maintained throughout the procedure. Heparin (5,000 units) was administered intravenously as a bolus prior to the procedure, the target coronary artery was engaged using standard 7 F guide catheters and control angiograms of both coronary arteries were performed using a nonionic contrast agent in two orthogonal views.

The pigs were randomized into two groups with 10 coronaries per group. One group received the control bare metal stents (3.0×18 mm, MAC®stent, AMG, Munich, Germany) and the second group received dual-coated stents (3.0×18 mm, abciximab and ALA coated MAC®stent). The stents were implanted in the proximal left anterior descending artery and proximal left circumflex artery and were deployed by inflating the balloon. The resulting stent-to-artery ratio was 1.3 : 1. Coronary angiograms were obtained immediately after stent implantation. All equipment was then removed and the carotid artery was ligated. All pigs continued to receive 100 mg of aspirin and 75 mg of clopidogrel daily until death.

Four weeks after stenting, the animals underwent follow-up angiography in the same orthogonal views. They were then sacrificed with an intercoronary injection of 20 mL of potassium chloride. The hearts were removed and the coronary arteries were pressure-perfusion fixed at 110 mmHg in 10% neutral buffered formalin overnight. The arteries were step-sectioned, processed routinely for light microscopy, and stained for histological analysis.

Histopathologic evaluation of each artery was performed by an experienced cardiovascular pathologist. The specimens were embedded in methylmethacrylate and sections were cut with a low-speed diamond wafer mounted to a Buehler Isomet saw (Buehler Ltd., Lake Bluff, IL, USA), leaving the stent wires intact in the cross sections to minimize potential artifacts caused by removal of the stent wires. Sections of 50 to 100 µm thickness were obtained at about 1 mm apart and stained with hematoxylin-eosin for histological analysis (Fig. 2). Measurements of the histopathologic sections were performed using a calibrated microscope, digital video imaging system, and microcomputer program (Visus 2000 Visual Image Analysis System, IMT Tech, CA, USA). Borders were manually traced for the lumen area, the 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. 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)}.

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.18) where 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.

The inflammation score for each individual strut was graded as follows: 0=no inflammatory cells surrounding the strut; 1=light, noncircumferential lymphohistiocytic infiltrate surrounding strut; 2=localized, moderate to dense cellular aggregate surrounding the strut noncircumferentially; and 3=circumferential dense lymphohistiocytic 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.19) Inflammatory cell counts were normalized to neointimal hyperplasia area and percent area stenosis was compared between the two stent groups. Ordinal data for fibrin were collected on each stent section using a scale of 0 to 3 as previously reported (Fig. 3).20)

Statistical analysis was performed with the aid of the commercially available software (Statistical Package for the Social Sciences Version 15, Chicago, IL, USA). The data are presented as mean value±SD. Unpaired Student's t-test was used for the comparison of inflammatory cell counts normalized to injury score of the two stent groups. Analysis of variance was used for comparisons of the two stent groups. To examine the correlations between the measured histologic variables, regression analysis was applied for each set of measured variables. A p<0.05 was considered statistically significant.