Vascular Balloon Injury and Intraluminal Administration in Rat Carotid Artery

Summary

This protocol uses a balloon catheter to cause an intraluminal injury on the rat carotid artery and henceforth elicit neointimal hyperplasia. This is a well-established model for studying the mechanisms of vascular remodeling in response to injury. It is also widely used to determine the validity of potential therapeutic approaches.

Abstract

The carotid artery balloon injury model in rats has been well established for over two decades. It remains an important method to study the molecular and cellular mechanisms involved in vascular smooth muscle dedifferentiation, neointima formation and vascular remodeling. Male Sprague-Dawley rats are the most frequently employed animals for this model. Female rats are not preferred as female hormones are protective against vascular diseases and thus introduce a variation into this procedure. The left carotid is typically injured with the right carotid serving as a negative control. Left carotid injury is caused by the inflated balloon that denudes the endothelium and distends the vessel wall. Following injury, potential therapeutic strategies such as the use of pharmacological compounds and either gene or shRNA transfer can be evaluated. Typically for gene or shRNA transfer, the injured section of the vessel lumen is locally transduced for 30 min with viral particles encoding either a protein or shRNA for delivery and expression in the injured vessel wall. Neointimal thickening representing proliferative vascular smooth muscle cells usually peaks at 2 weeks after injury. Vessels are mostly harvested at this time point for cellular and molecular analysis of cell signaling pathways as well as gene and protein expression. Vessels can also be harvested at earlier time points to determine the onset of expression and/or activation of a specific protein or pathway, depending on the experimental aims intended. Vessels can be characterized and evaluated using histological staining, immunohistochemistry, protein/mRNA assays, and activity assays. The intact right carotid artery from the same animal is an ideal internal control. Injury-induced changes in molecular and cellular parameters can be evaluated by comparing the injured artery to the internal right control artery. Likewise, therapeutic modalities can be evaluated by comparing the injured and treated artery to the control injured only artery.

Introduction

Balloon catheters are medical devices used in the procedure of angioplasty, for the purpose of widening obstructed site(s) of atheroma or thrombus in a blood vessel. The narrowed vessel lumen is forced to open up by the inflated balloon and blood supply would be restored sequentially to relieve downstream ischemia symptoms, such as angina, myocardial infarction, and leg pain. Nevertheless, the great success of angioplasty has been diminished by post-operative complications such as results from force causing vascular barotrauma (balloon injury), namely vessel wall remodeling and in many cases re-narrowing of the vessel lumen (restenosis)¹.

Protocol

NOTE: The use of animals for the following experiments has been reviewed and approved by the Institutional Animal Care and Use Committee (IACUC).

1. Preoperative Procedures

1. Sterilize surgical instruments before use.
   1. Autoclave all surgical instruments 24 hr or less before surgery. If multiple surgeries are performed on the same day, sterilize the instruments by a dry bead sterilizer in between surgeries.
2. Filter-sterilize the saline solution before use.
3. Weigh the rat and calculate the dose of anesthetic drugs (ketamine 80 mg/kg and xylazine 7 mg/kg).
4. Administer the anes....

Results

Two weeks after injury, carotid arteries are harvested, sectioned and subjected to morphological analysis. Arteries are cross-sectioned and stained with H&E (Figures 1, 2B, C and 3). Rat carotid artery wall contains four layers of elastic lamina, which appear as pink lines. The area between the outermost lamina, external elastic lamina (EEL) and the innermost lamina, internal elastic lamina (IEL) is the media smooth muscle layer (Figure 1). The area inside of the IEL is the intima, a.......

Discussion

The rat carotid balloon injury has been well described by Tulis in 2007³⁴. It has been comprehensively discussed all the details of this procedure by Dr. Tulis. The readers who are interested in performing this procedure are highly recommended to read Tulis’ protocol. However, there is one thing we are not agreeing with Dr. Tulis: Instead of inflating the balloon with saline or any kind of liquid, we suggested to inflate it with air. According to our personal experience, inflating with liquid can hardly avoid air bubbles. In.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Fogarty balloon embolectomy catheters, 2 French	Edwards Lifesciences, Germany	120602F
Deltaphase Operating Board - Includes 2 Pads & 2 Insulators	Braintree Scientific, Inc.	39OP
LED light source	Fisher Scientific	12-563-501
Hartmann Mosquito Forceps 4” curved	Apiary Medical, Inc. San Diego, CA	gS 22.1670
Crile Retractor 4” double ended	Apiary Medical, Inc.	gS 34.1934
Other surgical instruments	Roboz Surgical Instrument Company, Inc., Gaithersburg, MD
Peripheral Intravenous (I.V.) Cannula, 24 G	BD	381312
Ketamine HCl, 100 mg/ml, 10 ml	Ketaset- Patterson Vet	07-803-6637
Xylazine (AnaSed), 20 mg/ml, 20 ml	Ketaset- Patterson Vet	07-808-1947
Buprenex, 0.3 mg/1 ml (5 Ampules/Box)	Ketaset- Patterson Vet	07-850-2280
Nair Baby Oil Hair Removal Lotion - 9 oz	Amazon/Walmart/CVS	N/A
Inflation Device	Demax Medical	DID30
D300 3-way Stopcock	B.Braun Medical Inc.	4599543
Artificial Tears Ointment	Rugby Laboratories, Duluth, GA	N/A