The Rabbit Model of Accelerated Atherosclerosis: A Methodological Perspective of the Iliac Artery Balloon Injury

Summary

Animal models of atherosclerosis are essential to understand the mechanism and to investigate newer approaches to prevent plaque development or rupture, a leading cause of death in the industrialized world. This protocol uses a combination of balloon injury and cholesterol rich diet to induce atherosclerotic plaques in rabbit iliac artery.

Abstract

Acute coronary syndrome resulting from coronary occlusion following atherosclerotic plaque development and rupture is the leading cause of death in the industrialized world. New Zealand White (NZW) rabbits are widely used as an animal model for the study of atherosclerosis. They develop spontaneous lesions when fed with atherogenic diet; however, this requires long time of 4 - 8 months. To further enhance and accelerate atherogenesis, a combination of atherogenic diet and mechanical endothelial injury is often employed. The presented procedure for inducing atherosclerotic plaques in rabbits uses a balloon catheter to disrupt the endothelium in the left iliac artery of NZW rabbits fed with atherogenic diet. Such mechanical damage caused by the balloon catheter induces a chain of inflammatory reactions initiating neointimal lipid accumulation in a time dependent fashion. Atherosclerotic plaque following balloon injury show neointimal thickening with extensive lipid infiltration, high smooth muscle cell content and presence of macrophage derived foam cells. This technique is simple, reproducible and produces plaque of controlled length within the iliac artery. The whole procedure is completed within 20 - 30 min. The procedure is safe with low mortality and also offers high success in obtaining substantial intimal lesions. The procedure of balloon catheter induced arterial injury results in atherosclerosis within two weeks. This model can be used for investigating the disease pathology, diagnostic imaging and to evaluate new therapeutic strategies.

Introduction

Rupture of vulnerable atherosclerotic plaques is one of the leading causes of death in the industrialized nations¹. Although research over the past decades has unfolded several molecular and cellular mechanisms involved in plaque progression, continued efforts are still needed not only to unravel the complex mechanism of disease progression but also to test new therapeutic approaches. Several animal models have been proposed to study the atherosclerosis. Genetic manipulation, cholesterol feeding or mechanical endothelium injury are the standard strategies shared by most animal models of atherosclerosis including mice, rabbits or minipigs. Among these, NZW rabbits are sensitive to cholesterol diet while normal rats and mice do not significantly absorb dietary cholesterol^2,3,4. Rabbits spontaneously develop aortic lesions rich in macrophages with some fibrous component when fed with cholesterol rich diet^5,6. However, the long preparatory time of 4-8 months to induce atherosclerotic plaquesby feeding cholesterol diet alone^6,7 is a major drawback for most of the experimental settings. In pursuit for inducing lesions in relatively short time, a combination of high cholesterol diet and balloon injury has been developed by Baumgarter and Studer⁸. The overall goal of this technique is to induce atherosclerotic plaques composed of foam cells (similar to fatty streak in humans) in hypercholesterolemic rabbits within 2 weeks. The present technique describes the procedure of arterial wall injury based on Baumgarter's method using a balloon catheter advanced into the iliac artery of NZW hypercholesterolemic rabbits.

Together with a cholesterol rich diet, injury resulting from balloon induced de-endothelialization will lead to atherosclerosis. Balloon injury accelerates the formation of atherosclerotic lesions, and produces plaque of uniform size and distribution. Intimal thickening increases over a period of time and intimal cell infiltration starts within few days following injury. Fatty streaks with substantial macrophages start to appear after 7 - 10 days of balloon injury and are represented as Type II lesion according to the classification by American Heart Association. Balloon injury in rabbit is often performed in the aorta to study plaque composition. The neointimal endothelium expresses high levels of intercellular adhesion molecule. The plaques are associated with medial dissection and adventitial changes. Atherosclerotic lesions are composed of lipids, proliferating smooth muscle cells (SMCs), collagen fibers and inflammatory cells that accumulate under the regenerated endothelium and are mostly type II in nature. The topological distribution of rabbit plaques was similar to that reported in human aortas ^9,10 In principle, the aorta is larger in size compared to iliac arteries and would produce plaque in larger length. However, the major advantage of using the iliac artery as the site of atherosclerosis in rabbits is its accessibility, its similarity in muscular content to human coronary artery¹¹, uniform lesion development¹², high tissue factor activity¹³ and consistent vessel dimension comparable to human coronary artery allowing the evaluation of commercially manufactured devices to morphometric and angiographic endpoints. Invasive and non-invasive methods have been investigated to analyze the plaques in rabbit iliac arteries in the live animal. Previous reports describe the use of magnetic resonance imaging (MRI) with the help of a 2.35-tesla MR system ¹⁴ Additionally, intravascular ultrasound (IVUS) or optical coherence tomography (OCT) catheters can be suitably applied to image atherosclerotic plaques in rabbit iliac arteries. The iliac artery is accessible for ultrasound imaging when using a high-resolution echography and the aorta can also be explored with this technique.

In the past decade, this rabbit model of balloon injury has helped to further understand the mechanisms of plaque progression¹⁵and plaque regression¹⁶. In addition, the model has been used to study the influence of novel therapeutic agents such as statins, standard antiplatelet agents, antioxidant agents^17,18 and drug-eluting stents such as everolimus or zotarolimus-eluting stent^19,20 on neointimal thickening. This model has also been used to investigate intravascular imaging of near-infrared fluorescence imaging catheter²¹.

Protocol

This experimental protocol has been approved by the Cantonal Veterinary Office, Fribourg and the Swiss Federal Veterinary Office, Switzerland (FR 2015/58).

NOTE: Male NZW rabbits weighing between 2.8 to 3.2 kg were used. The animals were housed under conventional conditions (12 h light and dark cycle, provided ad libitum water and food). Prior to balloon denudation, animals were acclimated for 1 week during which they were fed with normal chow diet. After 1 week of acclimatization, rabbits were switched to atherogenic diet consisting of high fat (8.6%), and saturated fatty acids with 205 mg/kg cholesterol (1%) diet for the whole study duration. Balloon injury in the left iliac artery was performed 1 week after diet initiation and animals were sacrificed after 2 weeks or 4 weeks of balloon injury.

1. Preoperative Procedures

1. Sterilize all surgical instruments before use with a glass bead sterilizer or other suitable instrument.
2. Prepare and check the balloon catheter assembly.
   1. Attach a 1 mL luer lock syringe filled with normal saline to the luer-lock part of the balloon catheter. Carefully monitor for absence of trapped air. Check the leaks and ensure proper balloon inflation by pressing the plunger of the syringe.
3. Weigh the rabbit and turn on the thermopad to 37 °C.
4. Use a buprenorphine solution at a concentration of 0.3 mg/ml. Inject a dose of 0.01 mg/kg subcutaneously.
5. Anesthetize the rabbit with 5% isoflurane and 5 L/min O₂ in an induction chamber for 10 - 15 min.
6. Place the anesthetized rabbit on the heating pad kept on the surgical platform. Place the patch and clips to monitor the temperature, respiration, and electrocardiogram.
7. Attach the snout of the rabbit to a face mask connected to a suitable anaesthesia machine. Maintain the anaesthesia with isoflurane (4.0% with 2.5 L/min O₂). Confirm proper anesthetization (indicated by lack of muscle tone and loss of gag and pinnae reflexes).
8. Apply ophthalmic ointment to both eyes to prevent the corneas from drying. Drape the rabbit with a sterile surgical sheet with only the lower limb exposed.

2. Surgical Protocol

1. Remove the hair from the ventral area just below the knee joints using animal hair clippers.
2. Swab the area with suitable disinfectant to clean the skin and remove loose hair.
3. Locate the saphenous artery and make a small skin incision of about 1.5 cm in length using a scalpel.
4. Expose a small portion of saphenous artery with small curved forceps without damaging the femoral vein and femoral nerve.
5. Place two loose ligature loops (5-0 silk) beneath the saphenous artery and tie one ligature loop towards the distal end of the artery. Place a microvascular clamp above the ligature to stop the blood flow from the iliac artery.
6. Topically apply one drop of papaverine to dilate the artery and to prevent vasospasm.
7. Lift up the saphenous artery with the help of the tied ligature and make a small arteriotomy incision using a 24 gauge needle.
8. Elevate the incision flaps with fine forceps and slowly insert a vein pick or a guiding needle into the lumen of the artery.
9. Insert a 2 French Fogarty arterial embolectomy catheter into the saphenous artery. Remove the vein pick and microvascular clamps.
10. Advance the catheter till the sixth mark (20 - 25 cm) corresponding to a position roughly 2-5 cm above the iliac bifurcation.
11. Inflate the balloon with 0.1 mL normal saline using a 1 mL syringe or at a nominal pressure of 6 atm using a regulated manual inflator as described in ^16,22.
12. Hold the balloon catheter with forceps and drag back by 6 cm through the iliac arterytoward the point of insertion, while rotating the catheter.
13. Deflate the balloon by pulling back the plunger of the syringe.
14. Repeat steps 2.10 to 2.13 three times to ensure complete endothelial denudation.
15. Remove the catheter and immediately tie the ligature loop just above the arteriotomy site to stop bleeding.
16. Apply suitable antiseptic all around the periphery of the wound and swab away the blood clots. Close the skin incision with a 5-0 suture, and disinfect the surgery site with povidone-iodine solution.
17. Repeat steps 2.1 to 2.16 on the contralateral iliac using a new catheter.
18. Swab the ophthalmic ointment from eyes.

3. Post-operative Care

1. Administer sulfadoxine 40 mg/kg and trimethoprim 8 mg/kg or any other suitable antibiotic immediately after the surgical procedure.
2. During the anesthesia-recovery period, keep the rabbit over a heat pad placed in a clean autoclaved cage.
3. Remove the monitoring patch and clips.
4. After recovery, return the rabbits to their home cages. Inject subcutaneously buprenorphine 0.05 - 0.1 mg/kg post -operatively every 6 - 12 h for 48 h. Continue atherogenic diet for another two weeks or four weeks.

4. Tissue Harvesting and Analysis of Plaque Composition

1. After two weeks (for early thin plaque) or three weeks of balloon injury, anesthetize the rabbit using isoflurane in a similar way as described above.
2. Open the thoracic cavity and euthanize the rabbits by intracardial exsanguination.
3. Isolate the iliac arteries as described in ²³.
   1. Briefly, open the abdomen and expose the retroperitoneum. Trace the aorta towards the iliac bifurcation and tie it above the bifurcation. Carefully remove the surrounding tissues to expose and isolate both iliac arteries.
4. Dissect out both iliac arteries and immerse them in ice-cold phosphate buffered saline. Remove the clots with the help of forceps. Divide each iliac artery into 4 - 6 segments to characterise the thickness of the plaque throughout the artery.
5. Immediately embed the arterial segments in a mold containing optimum cutting temperature compound, snap-freeze using liquid nitrogen and keep it at -70 °C. Prepare 5 µm thick sections using a cryostat as described in ²⁴.
6. Perform histology, immunofluorescence or immunohistochemical staining for morphometry, plaque lipid and cellular content as described in ^10,25.
   NOTE: Briefly, rinse the arterial sections with phosphate buffered saline (PBS) and permeabilize using 0.2 % Triton. Rinse the sections with PBS and block non specific sites with 2% bovine serum albumin for 30 min. Incubate the sections for 1 h at 37 °C with anti α-SM actin (1:200) or RAM11 antibody (1:200). Rinse the sections with PBS and incubate them with appropriate secondary antibody for 30 min at 37 °C. Wash again with PBS and add Hoechst (5 µg/mL) for 10 min to detect nuclei.

Results

Balloon injury of the iliac artery was performed successfully without complication (Figure 1). The total operative time ranged from 20 to 30 min for injuries performed on only one iliac artery, and 35 to 45 min for injuries on both arteries. The rabbit recovered within 1 h after balloon injury. All animals appeared healthy without significant weight loss. No infection, oedema or arterial thrombosis was encountered. The wound area was normal besides some mild ...

Discussion

The rabbit iliac artery atherosclerosis model is widely used in atherosclerosis research. With this protocol the rabbits rapidly developed more severe and advanced plaques as compared to spontaneous lesions developed with only cholesterol diet. Importantly, animals recover quickly from the surgery.

The main stimulus for atherogenesis is the mechanical damage caused by the balloon catheter that injures the endothelium and distends the vessel wall²⁶. This procedure induce...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
New Zealand White rabbits	Charles River laboratories,France	Cre:KBL(NZW)
Cholesterol rich diet	Ssniff spezialdiäten	Ssniff EF K High Fat and Cholesterol
Glass bead sterilizer-Germinator 500	VWR, Leicestershire, UK	101326-488
Fogarty balloon embolectomy catheters, 2 French	Edwards Lifesciences, Switzerland	120602F	For single use only
Luer Lock Syringe	Becton, Dickinson and Company, USA	309628
Thermopad Type 226	Solis, Switzerland AG	397387
Buprenorphine- Temgesic	Reckitt Benckiser AG, Switzerland	7.68042E+12
Isoflurane	Piramal Critical Care, Inc, Bethlehem, PA 18017	2667-46-7
Anaesthesia machine-combi-vet Base Anesthesia System	Rothacher Medical GmbH, Switzerland	CV 30-301-A
Cardell touch veterinary vital signs monitor	Midmark, Ohio, USA	8013-001
Ophthalmic ointment-Humigel	Virbac, France
Animal hair clippers	Aesculap AG, Germany	GT420
Disinfectant-Betadine solution	MundipharmaMedicalCompany, Switzerland	14671-1203
Dumont #7 Forceps	FST Germany	11274-20
Medium and small microscissors	Medline International Switzerland Sàrl	UC4337
Microvascular clamps	FST, Germany	18051-28
Papaverine	ESCA chemicals, Switzerland	RE 356 803
Vein Pick	Harvard Apparatus, Cambridge, UK	72-4169	For single use only
Saline	Laboratorium Dr. G. Bichsel AG, , Switzerland	1330055
Polysorb 5-0 suture	Covidien AG, Switzerland	UL 202	Monofilament
Sulfadoxine and Trimethoprim-Trimethazol	Werner Stricker AG, Switzerland	Swissmedic Nr. 50'361
Antiseptic- Octenisept	Schülke & Mayr AG, Switzerland	GTIN: 4032651214068
Phosphate Buffered Saline	Roth	1058.1
Isobutanol-2-Methylbutane	Sigma-Aldrich, Switzerland	M32631-1L
Optimum Cutting Temperature compound-Tissue-Tek	VWR Chemicals, Belgium	25608-930
Cryostat	Leica, Glattbrugg, Switzerland	Leica CM1860 UV
Glass slide- Superfrost Plus	Thermo Scientific	4951PLUS4
Mayer's Haematoxylin	Sigma-Aldrich, Switzerland	MHS32-1L
Eosin 0.5% aq.	Sigma-Aldrich, Switzerland	HT110232-1L
Oil Red O	Sigma-Aldrich, Switzerland	O0625-25G
α-smooth muscle actin antibody	Abcam, UK.	ab7817
Macrophage Clone RAM11 antibody	DAKO, Switzerland	M063301
Hoechst	Abcam, UK.	ab145596
Goat polyclonal Secondary Antibody (Chromeo 546)	Abcam, UK.	ab60316
Alexa Fluor 488/547	Abcam, UK.
Glycergel Mounting Medium, Aqueous	DAKO, Switzerland	C056330
Hematoxylin for Movat pentachrome staining	Sigma-Aldrich, Switzerland	H3136-25G
Ferric chloride for Movat pentachrome staining	Sigma-Aldrich, Switzerland	157740-100G
Iodine for Movat staining	Sigma-Aldrich, Switzerland	207772-100G
Potassium iodide for Movat pentachrome staining	Sigma-Aldrich, Switzerland	60400-100G-F
Alcian blue for Movat staining	Sigma-Aldrich, Switzerland	A5268-10G
Strong Ammonia for Movat pentachrome staining	Sigma-Aldrich, Switzerland	320145-500ML
Brilliant crocein MOO for Movat pentachrome staining	Sigma-Aldrich, Switzerland	210757-50G
Acid Fuchsin for Movat pentachrome staining	Sigma-Aldrich, Switzerland	F8129-50G
Sodium Thiosulfate for Movat pentachrome staining	Sigma-Aldrich, Switzerland	72049-250G
Phosphotungstic acid for Movat pentachrome staining	Sigma-Aldrich, Switzerland	79690-100G
Crocin for Movat pentachrome staining	Sigma-Aldrich, Switzerland	17304-5G
EUKITT for Movat pentachrome staining	Sigma-Aldrich, Switzerland	03989-100ML