**2. Methods**

Three male New Zealand white rabbits (3.5–4 kg) were obtained from the Charles River Laboratories, France. The animals were housed in the animal centre facility at the University of Fribourg (Switzerland). All animals received humane care in compliance with the European Convention on Animal Care and in accordance with the Swiss Animal Protection Law after obtaining permission from the State Veterinary Office, Fribourg approved by the Swiss Federal Veterinary Office, Switzerland (FR-2016/16).

Angioplasty: Under general anaesthesia induced with s.c. injection of Narketan (65 mg/ml) and Xylapan (4 mg/ml) and maintained by perfusion (Narketan 65 mg/50 ml, Xylapan 4 mg/50 ml, infusion 15–20 ml/h), heparin (100 UI/ml) was administered in the marginal vein using a 24 GA (BD Insyte) catheter. Body parameters, including temperature, heart rate and pO2 were controlled by a veterinary monitor (Midmark Cardell touch). In clean condition, an arteriotomy of the left coronary artery was performed, and a 6-French introducer sheath (Glidesheath Slender, Terumo) positioned. Two ml of a contrast agent (Bracco, Iomeprol 35 g) was injected, and a 0.36-mm guidewire is advanced through an introducer sheath up to the right iliac artery. Under angiographic monitoring of the pelvic area (**Figure 1**), the stent from Baxter (Coroflex Blue Neo) was deployed and overexpanded. The balloon was then retracted and then directed within the contralateral iliac artery (left), positioned at the same distance from the aortic bifurcation and inflated at 10 atm for 30 seconds to induce endothelial injury. At the end of the procedure, s.c. injection of Temgésic (1 ml/kg), Trimethazol (Werner Stricer, composed of sulfadoxine 40 mg/kg and trimethoprim 8 mg/kg) and carprofen 2.2 mg/ kg (Rimadyl, Zoetis) were performed. The rabbit awoke within 1 h. Animals were kept on a normal diet for up to 8 weeks.

High-resolution ultrasound image acquisitions were performed every week under general gaze anaesthesia induced by isoflurane 4–5% and O2 2–3 L/h. The animals were placed on an in-house-made platform, on a heating pad. The animal temperature was monitored through the rectal probe provided with the Vevo3100. ECG stainless steel needle electrodes provided with the Vevo3100 were placed

**185**

**Figure 1.**

*of the stent placement.*

*High-Resolution Ultrasound Imaging System for the Evaluation of the Vascular Response to Stent…*

subcutaneously on the four limbs (right and left upper, right and left lower). The respiration rate was derived from the ECG signal. Longitudinal analyses were performed with a Vevo3100, VisualSonic high-resolution ultrasound system equipped with a transducer MX400 (20–46 Mhz) hold with the imaging station arm. The images were acquired at day 0, 14, 28, 42 and 56 or 63 in all animals. The image analyses were performed with the VevoVasc analysis module. The following parameters were quantified: wall thickness, lumen diameter and cross-sectional area (CSA). Using a Doppler pulse-wave mode, the velocity-time integral (VTI) was extracted. The following calculation was performed with the measured parameters: the blood flow was calculated as the product of the VTI and the CSA of the respective segments. The percentage changes in parameters relative to the values before the intervention were calculated as a ratio of the parameter at day 14, 28 or 42 to the

*Bilateral iliac artery injuries performed by an inflated balloon (left artery) and stent overexpansion (right artery). (A) Diagram showing the positioning of the stent and contralateral balloon placement. Each animal received a single stent that was placed either close to the aortic bifurcation or distally. (B) Angiography of both iliac arteries: The stent was placed first in the right artery and overexpanded, and the balloon was then retracted back to the aorta and introduced into the left artery. Balloon inflation was performed at a similar distance from the aortic bifurcation. S, stent; B, balloon. (C) Illustration of the positioning of the stent: 8 weeks post-intervention, the right iliac was exposed, and the transmural visualisation of the stent confirmed the site* 

Artery harvesting was performed after euthanasia. Distal aortic segment and both iliac arteries were harvested according to the well-established procedure [10]

Histology characterisation: OCT embedding the vessel segments was frozen in the vapour of 2-methylbutane placed in liquid nitrogen. Sections of 5 μm are obtained using a cryocut and were processed for Movat Pentachrome staining. Briefly, sections were fixed 1 h in Bouin for 56°C, stained with Alcian Blue followed by Verhoeff's Elastic Stain, differentiated in ferric chloride solution, stained in brilliant crocein 1% and acid fuchsin 1%, placed in 5% phosphotungstic acid and

Stented segments were embedded in epoxy. 0.8-μm sections were cut with an

same parameter at day 0 and multiplied by 100.

and cut in short segments for histological analysis.

ultramicrotome and stained with methyl blue.

stained with crocin. Sections were mounted with EUKITT®.

*DOI: http://dx.doi.org/10.5772/intechopen.88656*

*High-Resolution Ultrasound Imaging System for the Evaluation of the Vascular Response to Stent… DOI: http://dx.doi.org/10.5772/intechopen.88656*

#### **Figure 1.**

*Animal Models in Medicine and Biology*

first generation of the bare-metal stents (BMS, permanent metallic structure without drug release) to the last generation of drug-eluting stents (DES, permanent metallic structure with anti-proliferative drug release). The BMS and DES are mostly made of a cobalt-chromium alloy and remain lifelong in the artery of the patient. Several studies showed, however, that life-threatening complication, emerging several months or years after implantation, may occur, including restenosis due to neointimal hyperplasia and late in-stent thrombosis [2]. Novel therapeutic approaches to reduce persistent inflammation, stenosis and thrombosis are focused on antiproliferative and anti-inflammatory processes such as drug-eluting stents [3], pharmaceutical [4, 5] or laser-based approaches [6, 7] as well as bioresorbable stents [8]. In this context, an appropriate animal model is paramount to foster the development of new therapies, to provide in vivo preclinical proof of concept, to evaluate the treatment performance and to promote translation to the clinic. The rabbitinjured iliac artery model has been well established to investigate the vascular

In the present study, we evaluated the vascular responses to bilateral iliac artery injuries performed by balloon denudation and stent overexpansion, using a highresolution ultrasound imaging system. We explored the longitudinal evolution of

Three male New Zealand white rabbits (3.5–4 kg) were obtained from the Charles River Laboratories, France. The animals were housed in the animal centre facility at the University of Fribourg (Switzerland). All animals received humane care in compliance with the European Convention on Animal Care and in accordance with the Swiss Animal Protection Law after obtaining permission from the State Veterinary Office, Fribourg approved by the Swiss Federal Veterinary Office,

Angioplasty: Under general anaesthesia induced with s.c. injection of Narketan

High-resolution ultrasound image acquisitions were performed every week under general gaze anaesthesia induced by isoflurane 4–5% and O2 2–3 L/h. The animals were placed on an in-house-made platform, on a heating pad. The animal temperature was monitored through the rectal probe provided with the Vevo3100. ECG stainless steel needle electrodes provided with the Vevo3100 were placed

(65 mg/ml) and Xylapan (4 mg/ml) and maintained by perfusion (Narketan 65 mg/50 ml, Xylapan 4 mg/50 ml, infusion 15–20 ml/h), heparin (100 UI/ml) was administered in the marginal vein using a 24 GA (BD Insyte) catheter. Body parameters, including temperature, heart rate and pO2 were controlled by a veterinary monitor (Midmark Cardell touch). In clean condition, an arteriotomy of the left coronary artery was performed, and a 6-French introducer sheath (Glidesheath Slender, Terumo) positioned. Two ml of a contrast agent (Bracco, Iomeprol 35 g) was injected, and a 0.36-mm guidewire is advanced through an introducer sheath up to the right iliac artery. Under angiographic monitoring of the pelvic area (**Figure 1**), the stent from Baxter (Coroflex Blue Neo) was deployed and overexpanded. The balloon was then retracted and then directed within the contralateral iliac artery (left), positioned at the same distance from the aortic bifurcation and inflated at 10 atm for 30 seconds to induce endothelial injury. At the end of the procedure, s.c. injection of Temgésic (1 ml/kg), Trimethazol (Werner Stricer, composed of sulfadoxine 40 mg/kg and trimethoprim 8 mg/kg) and carprofen 2.2 mg/ kg (Rimadyl, Zoetis) were performed. The rabbit awoke within 1 h. Animals were

response to hyperplasia and stenosis or thrombosis [3, 8, 9].

the vessel morphometries and the blow flow.

**2. Methods**

Switzerland (FR-2016/16).

kept on a normal diet for up to 8 weeks.

**184**

*Bilateral iliac artery injuries performed by an inflated balloon (left artery) and stent overexpansion (right artery). (A) Diagram showing the positioning of the stent and contralateral balloon placement. Each animal received a single stent that was placed either close to the aortic bifurcation or distally. (B) Angiography of both iliac arteries: The stent was placed first in the right artery and overexpanded, and the balloon was then retracted back to the aorta and introduced into the left artery. Balloon inflation was performed at a similar distance from the aortic bifurcation. S, stent; B, balloon. (C) Illustration of the positioning of the stent: 8 weeks post-intervention, the right iliac was exposed, and the transmural visualisation of the stent confirmed the site of the stent placement.*

subcutaneously on the four limbs (right and left upper, right and left lower). The respiration rate was derived from the ECG signal. Longitudinal analyses were performed with a Vevo3100, VisualSonic high-resolution ultrasound system equipped with a transducer MX400 (20–46 Mhz) hold with the imaging station arm. The images were acquired at day 0, 14, 28, 42 and 56 or 63 in all animals. The image analyses were performed with the VevoVasc analysis module. The following parameters were quantified: wall thickness, lumen diameter and cross-sectional area (CSA). Using a Doppler pulse-wave mode, the velocity-time integral (VTI) was extracted. The following calculation was performed with the measured parameters: the blood flow was calculated as the product of the VTI and the CSA of the respective segments. The percentage changes in parameters relative to the values before the intervention were calculated as a ratio of the parameter at day 14, 28 or 42 to the same parameter at day 0 and multiplied by 100.

Artery harvesting was performed after euthanasia. Distal aortic segment and both iliac arteries were harvested according to the well-established procedure [10] and cut in short segments for histological analysis.

Histology characterisation: OCT embedding the vessel segments was frozen in the vapour of 2-methylbutane placed in liquid nitrogen. Sections of 5 μm are obtained using a cryocut and were processed for Movat Pentachrome staining. Briefly, sections were fixed 1 h in Bouin for 56°C, stained with Alcian Blue followed by Verhoeff's Elastic Stain, differentiated in ferric chloride solution, stained in brilliant crocein 1% and acid fuchsin 1%, placed in 5% phosphotungstic acid and stained with crocin. Sections were mounted with EUKITT®.

Stented segments were embedded in epoxy. 0.8-μm sections were cut with an ultramicrotome and stained with methyl blue.

The Bersoft Image Analysis software (Bersoft Technology and Software; Lunenburg, Canada) was used to quantify the vessel diameter and the wall thickness (including intimal, media and adventitia layers).
