A Rat Carotid Artery Pressure-Controlled Segmental Balloon Injury with Periadventitial Therapeutic Application
Summary
The rat carotid artery balloon injury mimics the clinical angioplasty procedure performed to restore blood flow in atherosclerotic vessels. This model induces the arterial injury response by distending the arterial wall, and denuding the intimal layer of endothelial cells, ultimately causing remodeling and an intimal hyperplastic response.
Abstract
Cardiovascular disease remains the leading cause of death and disability worldwide, in part due to atherosclerosis. Atherosclerotic plaque narrows the luminal surface area in arteries thereby reducing adequate blood flow to organs and distal tissues. Clinically, revascularization procedures such as balloon angioplasty with or without stent placement aim to restore blood flow. Although these procedures reestablish blood flow by reducing plaque burden, they damage the vessel wall, which initiates the arterial healing response. The prolonged healing response causes arterial restenosis, or re-narrowing, ultimately limiting the long-term success of these revascularization procedures. Therefore, preclinical animal models are integral for analyzing the pathophysiological mechanisms driving restenosis, and provide the opportunity to test novel therapeutic strategies. Murine models are cheaper and easier to operate on than large animal models. Balloon or wire injury are the two commonly accepted injury modalities used in murine models. Balloon injury models in particular mimic the clinical angioplasty procedure and cause adequate damage to the artery for the development of restenosis. Herein we describe the surgical details for performing and histologically analyzing the modified, pressure-controlled rat carotid artery balloon injury model. Additionally, this protocol highlights how local periadventitial application of therapeutics can be used to inhibit neointimal hyperplasia. Lastly, we present light sheet fluorescence microscopy as a novel approach for imaging and visualizing the arterial injury in three-dimensions.
Introduction
Cardiovascular disease (CVD) remains the leading cause of death worldwide1. Atherosclerosis is the underlying cause of most CVD-related morbidity and mortality. Atherosclerosis is the build-up of plaque inside arteries that results in a narrowed lumen, hindering proper blood perfusion to organs and distal tissues2. Clinical interventions for treating severe atherosclerosis include balloon angioplasty with or without stent placement. This intervention involves advancing a balloon catheter to the site of plaque, and inflating the balloon to compress the plaque to the arterial wall, widening the luminal area. This procedure damages the artery, however, initiating the arterial injury response3. Prolonged activation of this injury response leads to arterial restenosis, or re-narrowing, secondary to neointimal hyperplasia and vessel remodeling. During angioplasty the intimal layer is denuded of endothelial cells leading to immediate platelet recruitment and local inflammation. Local signaling induces phenotypic changes in vascular smooth muscle cells (VSMC) and adventitial fibroblasts. This leads to the migration and proliferation of VSMC and fibroblasts inwards to the lumen, leading to neointimal hyperplasia4,5. Circulating progenitor cells and immune cells also contribute to the overall volume of restenosis6. Where applicable, drug-eluting stents (DES) are the current standard for inhibiting restenosis7. DES inhibit arterial re-endothelialization, however, thus creating a pro-thrombotic environment that can result in late in-stent thrombosis8. Therefore, animal models are integral for both understanding the pathophysiology of restenosis, and for developing better therapeutic strategies to prolong the efficacy of revascularization procedures.
Several large and small animal models9 are utilized for studying this pathology. These include balloon-injury3,10 or wire-injury11 of the luminal side of an artery, as well as partial ligation12 or cuff placement13 around the artery. The balloon and wire injury both denude the endothelial layer of the artery, mimicking what occurs clinically after angioplasty. In particular, balloon-injury models utilize similar tools as in the clinical setting (i.e., balloon catheter). The balloon injury is best performed in rat models, as rat arteries are an appropriate size for commercially available balloon catheters. Herein we describe a pressure-controlled segmental arterial injury, a well-established, modified version of the rat carotid artery balloon injury. This pressure-controlled approach closely mimics the clinical angioplasty procedure, and allows for reproducible neointimal hyperplasia formation two weeks after injury14,15. Additionally, this pressure-controlled arterial injury results in complete endothelial layer restoration by 2 weeks after surgery16. This directly contrasts the original balloon injury model, described by Clowes, where the endothelial layer never returns to full coverage3.
After surgery, therapeutics may be applied to or directed towards the injured artery through several approaches. The method described herein uses periadventitial application of a small molecule embedded in a Pluronic gel solution. Specifically, we apply a solution of 100 μM cinnamic aldehyde in 25% Pluronic-F127 gel to the artery immediately after injury to inhibit neointimal hyperplasia formation15. Pluronic-F127 is a non-toxic, thermo-reversible gel able to deliver drugs locally in a controlled manner17. Meanwhile, arterial injury is local, hence local administration allows for testing an active principle while minimizing off-target effects. Nevertheless, effective delivery of a therapeutic using this method will depend on the chemistry of the small molecule or biologic used.
Protocol
Representative Results
Discussion
The rat carotid artery balloon injury is one of the most extensively used and studied restenosis animal models. Both the original balloon injury model3 and the modified pressure-controlled segmental injury variation10 have informed many aspects of the arterial injury response that also occurs in humans, with the few limitations being that fibrin-rich thrombus rarely develops and local inflammation is minimal compared to other injury models such as in hypercholesterolemic ra…
Disclosures
The authors have nothing to disclose.
Acknowledgements
N.E.B. was supported by a training grant from the National Institute of Environmental Health Sciences (5T32ES007126-35, 2018), and an American Heart Association pre-doctoral fellowship (20PRE35120321). E.S.M.B. was a KL2 scholar partially supported by the UNC Clinical and Translational Science Award-K12 Scholars Program (KL2TR002490, 2018), and the National Heart, Lung, and Blood Institute (K01HL145354). The authors thank Dr. Pablo Ariel of the UNC Microscopy Services Laboratory for assisting with LSFM. Light Sheet Fluorescence Microscopy was performed at the Microscopy Services Laboratory. The Microscopy Services Laboratory, Department of Pathology and Laboratory Medicine, is supported in part by P30 CA016086 Cancer Center Core Support Grant to the UNC Lineberger Comprehensive Cancer Center.
Materials
| 1 mL Syringe | Fisher | 14955450 | |
| 1 mL Syringe with needle | BD | 309626 | |
| 2 French Fogarty Balloon Embolectomy Catheter | Edwards LifeSciences | 120602F | |
| 4-0 Ethilon (Nylon) Suture | Ethicon Inc | 662H | |
| 4-0 Vicryl Suture | Ethicon Inc | J214H | |
| 7-0 Prolene Suture | Ethicon Inc | 8800H | |
| 70% ethyl alcohol | |||
| Anti-Rabbit Alexa Fluor 647 | Thermo Fisher Scientific | A21245 | |
| Atropine Sulfate | Vedco Inc | for veterinary use | |
| Cotton Swabs | Puritan | 806-WC | |
| Curved Hemostats | Fine Science Tools | 13009-12 | |
| Fine Curved Forceps | Fine Science Tools | 11203-25 | |
| Fine Scissors | Fine Science Tools | 14090-11 | |
| Gauze | Covidien | 2252 | |
| IHC-Tek Diluent (pH 7.4) | IHC World | IW-1000 | |
| Insufflator | Merit Medical | IN4130 | |
| Iodine solution | |||
| Lubricating Eye Ointment | Dechra | for veterinary use | |
| Mayo Scissors | Fine Science Tools | 14010-15 | |
| Micro Serrefines | Fine Science Tools | 18055-05 | |
| Microdissection Scissors | Fine Science Tools | 15004-08 | |
| Micro-Serrefine Clamp Applying Forceps | Fine Science Tools | 18057-14 | |
| Needle Holder | Fine Science Tools | 12003-15 | |
| Pluronic-127 (diluted in sterile water) | Sigma-Aldrich | P2443 | 25% prepared |
| Rabbit Anti-CD31 | Abcam | ab28364 | |
| Retractor | Bent paper clips work well | ||
| Rimadyl (Carprofen) | Zoetis Inc | for veterinary use | |
| Saline solution | |||
| Standard Forceps | Fine Science Tools | 11006-12 | |
| Sterile Drape | Dynarex | 4410 | |
| T-Pins |
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