Histopathological evaluation of a retinoic acid eluting stent in a rabbit iliac artery model – Scientific Reports

The study was carried out in compliance with the ARRIVE guidelines24 and the European Union legislation25 following the principle of 3Rs (Reduction, Refinement, Replacement) to ensure: a reduction in the number of animals used, the improvement of experimental techniques and their living conditions so that they are kept to a minimum pain or suffering. The experiments were performed at the Biomedical Research Foundation of the Academy of Athens, Greece / Athens. The protocol was approved by the Protocol Evaluation Committee of the Biomedical Research Foundations of the Academy of Athens, Greece and the Directorate of Agricultural and Veterinary Policy of the Prefecture of Attica, Greece, a State authority. All methods were performed in accordance with the relevant National and international guidelines and regulations.

Stent placement

Endovascular stents were implanted in the iliac arteries of 30 male New Zealand (2.5 to 3.5 kg) rabbits. The rabbit iliac artery model was chosen as a first test for the preclinical evaluation of the RA eluting stents because the size and injury response of rabbit iliac arteries are relatively comparable to human coronary arteries22,26. The animals were individually housed in stainless steel cages. Two stents of the same group were implanted in each rabbit, one stent in each iliac artery. Sixty stents were used in total, which were divided in ten groups, six stents per group, three animals per group. The observation period after implantation was 4 weeks.

Stent implantation procedure

The rabbits were anesthetized by an intramuscular injection of ketamine (35 mg/kg) and xylazine (7 mg/kg) and were monitored by peripheral pulse oximetry during the procedure. We used a published method of percutaneous transauricular endovascular access27. In brief, the animals were placed in supine position, and immobilized. Both auricular dorsa were shaved and sterilized with povidone-iodine. A novice trainee and an experienced interventional cardiologist performed the trans-auricular vascular access, elective vessel catheterizations, and stent implantation in the iliac arteries.

For the initial implantations and in order to establish an accurate stent placement, as well as for collecting data required by a parallel study aiming to develop an in silico simulation of the expansion process28 optical coherence tomography (OCT) was performed post-procedural in a small number of stents (Ν = 4). The preparation and materials used for the process were slightly modified for those cases that OCT was performed.

The central auricular artery was punctured with a 22-gauge intravenous catheter approximately at the distal half of its subcutaneous course. Then the central needle of the catheter was removed. A 0.018-inch (V18, Boston scientific, MA) hydrophilic guide wire was carefully advanced into the external carotid artery and aortic arch / descending aorta and then into the thoracic aorta. The catheter was removed, and a 3-cm-long incision of the dermis was performed at the point of the initial puncture along the course of the guide wire to the base of the dorsa. In cases where OCT was scheduled to be performed, a 5-F compatible 0.035-inch, slender introducer sheath (Terumo, Tokyo, Japan) was advanced into the external carotid artery after serial step-by-step dilations with the sheath’s own dilator. Repeated over-the-wire dilations were performed in order to remove the tight and narrow peripheral segment of the artery prior to sheath insertion. Then, heparin (100 IU/kg) was administered intra-arterially. Upon access gaining a 5F guiding catheter (Envoy MPC, Codman, USA) was advanced to the distal aorta to get direct negotiation with targeting vessels and an angiography was performed to visually evaluate the vessel diameter and as roadmap for accessing femoral vessels. Following angiography, the wire was exchanged to an 0.014-inch wire (Lotus II, Rontis, Switzerland), and it was advanced to the femoral artery. Finally, the stent was forwarded and placed, a few millimeters after the origin of the iliac artery (10Atm, 20 s). Following the stent implantation, OCT was performed without contrast media but via washing out with saline solution only.

In cases where the OCT was not scheduled, the process was exactly the same as above, up to the point of the introducer sheath use entry. A 4-F, 0.018-inch compatible Radifocus Introducer II sheath (Terumo, Tokyo, Japan) was used to gain the access point and the following activities were the same as described above. Another differentiation was on the guiding catheter where the above referred catheter was replaced with a 4-F angiographic catheter (Impress VER-H, Merit Medical, USA). A final angiography was performed for evaluating the stent apposition, the vascular patency and the presence of possible complications (e.g.: formation of thrombus, presence of intimal flap, a sub-optimally deployed stent etc.). The animals did not receive any antiplatelet medication.

Type of stents

For the preparation of the drug eluting stents, Cobalt-Chrome bare metal stents (Leader Plus, Rontis, Zug, Switzerland) with dimensions 12 mm length × 2.50 mm diameter were coated with coatings consisting of a mixture of polymer and drug (Table 3). Two different types of polymers were used, polymer “A”, poly (lactic-co-glycolic acid) (PLGA) and polymer “B”, poly lactic acid (PLA). Three different doses of the drug RA were used with polymers “A” and “B” resulting in three groups of stents for each polymer: AL, AM, AH with polymer A and “Low”, “Medium” and “High” dose respectively and BL, BM, BH with polymer B and “Low”, “Medium” and “High” dose respectively. As retinoic acid is light and oxygen sensitive, care was given to protect the drug from degradation during the coating process and storage. The drug was handled under a dim yellow light during preparation of the coating mixtures. After coating and crimping on catheters, the stent systems were packaged in non-transparent aluminum foil packages with moisture and oxygen absorbers. In order to ensure the quantity of the drug on the stents, quality control tests were performed after production. Two groups of stents, namely EF and EG with the same dose of everolimus were prepared with the use of polymers A and B respectively. All coated stents were crimped on Rontis’ Leader Plus stent delivery systems with dimensions 2.5 × 12 (REF: LP20/12).

Table 3 The types of stents, compositions of the coatings and the drug doses of the tested items.

Rontis Cobalt-Chrome bare metal (uncoated) stents and commercially available Everolimus eluting stents (Xience Pro Everolimus Eluting Coronary Stent System, Abbot Vascular, Santa Clara CA, USA) were also used of the same size (group C and D, respectively). The types of stents, compositions of the coatings and the drug doses of the tested items are included in Table 3.

Necropsy and histomorphometry

The animals were euthanized with sodium pentobarbitone injection. Necropsies were performed by an experienced veterinary doctor. The treated artery segments were collected and preserved in 10% neutral buffered formalin and transferred for histological processing, image analysis and histopathological evaluation. Histology and morphometry were performed at an independent Good Laboratory Practice certified laboratory (Anapath Services, GmbH, Switzerland) by a board-certified veterinarian pathologist. Samples from implants sites were processed by methyl methacrylate (MMA) resin embedding, sawed by a diamond band saw in transversal sample direction (EXAKT System), ground and polished to a final thickness of approximately 40–60 µm (EXAKT System). One section from each sample was stained with adapted Paragon stain following Standard Operating Procedures. Evaluation of the slides for quality check was performed, and then slides were transferred to the study pathologist for pathology evaluation.

Evaluation parameters

The parameters evaluated by histopathology examination adapted to the scoring system described by ISO 10993-6:2016 were: arterial reaction [endothelial loss (%), surface (fibrin/platelet thrombus), intima proliferation, smooth muscle in intima proliferation, proteoglycan/collagen], artery inflammation (intima/media, Adventitia), medial smooth muscle cell (SMC) loss [Medial SMC loss (transmural), Medial SMC loss (circumference)], artery cell replacement tissue (proteoglycans, collagen, adventitia), host reaction associated with the implant (polymorphonuclear cells, lymphocytes, plasma cells, macrophages, giant cells, necrosis, fibrosis, peristrut hemorrhage/fibrin accumulation, neovascularization, fatty Infiltrate), elastic lamina (EL) rupture (external EL rupture, internal EL rupture), medial hypertrophy (focal, diffuse). An adapted scoring system, described by ISO 10993-6:2016, was applied. Each parameter scored from 0 to 4 according to the histological findings. Defined by the ISO 10993-6:2016, a score difference between 0.0 to 2.9 is considered no or minimal host reaction, 3.0 to 8.9 slight host reaction, 9.0 to 15.0 moderate host reaction and ≥ 15.1 severe host reaction compared to a reference material.

An Olympus Slideview VS200 slides scanner using an Olympus U-TV1XC camera and 20× objective was used to scan the arterial samples. Quantitative evaluation was performed using Olympus imaging and image analysis software cellSens v1.18.

Quantitative evaluation by image analysis on each artery was performed, including the following parameters: Area within external elastic lamina (EEL; µm2), area within internal elastic lamina (IEL; µm2), lumen (µm2), intima (µm2) (calculation: IEL-lumen), media (µm2) (calculation: EEL − IEL), stenosis (%) [calculation:100 − (100 × Lumen/IEL)], intimal thickness (µm) (average value from 10 approximate equidistant thick measurements). These arithmetic mean values were used for further descriptive statistics. Illustrative images with the measurements, one for each group, are displayed in Fig. 3. As arteries were not flushed during sampling to avoid missing any possible presence of local thrombosis, red blood cells (round anuclear cells) were present in the lumens of several arteries.

Figure 3
figure 3

Representative images displaying measurements, one image per treatment group. Paragon, objective ×20. The images show measurement parameters, external elastic lamina (EEL), internal elastic lamina (IEL), lumen, intima and media. Ten approximate equidistant measurements were used for measuring the intimal thickness. A Polymer A; PLG poly (lactic-co-glycolic acid); B polymer B; PLA poly lactic acid; L low (dose); M medium (dose), H high (dose); C Rontis bare metal stent; D Xience Pro everolimus eluting coronary stent system, Abbot vascular; EF and EG Rontis everolimus stents with the use of polymers A and B respectively. RA retinoic acid.

Statistical analysis

Statistical tests were performed using the Prism 8 software (GraphPad, San Diego, CA, USA). Descriptive statistics were used for medial area, intimal area, stenosis and intimal thickness. The Shapiro–Wilk test for normality was performed. When the data followed normal distribution, the comparisons were performed with the unpaired t-test. When data did not follow normal distribution, the Mann–Whitney test was used. A p value less than 0.05 was considered significant.

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