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.

A number of animal models have been developed mimicking the angioplasty procedure to help investigators understand mechanisms underlying the balloon-injury-related vessel wall remodeling². Among all the animal species utilized for modeling, rat is the most frequently used one. Compared to rabbits, dogs and swine, the advantages of rats are their low cost, their relative ease of use and the current knowledge of rat physiology. Although mice have an added advantage in a wide range of genetically manipulated strains, the mice vessel is too small to insert a balloon catheter. Over the past three decades, experimental rats have allowed researchers to gain better understanding of the molecular and cellular mechanisms underpinning neointima formation and vascular remodeling^3-6. Beyond balloon injury, vascular remodeling are also involved in most major vascular diseases, such as atherosclerosis^7,8, hypertension⁹, and aneurysm¹⁰. Thus, knowledge gained through the balloon injury model is in general beneficial to overall vascular wall disease studies.

The overall goal of the rat balloon injury model is not only to further understand vascular diseases but also to test the potency of novel agents for disease control^11,12. Current clinical drug treatment to restenosis is applied by drug-eluting stents placed via the vessel lumen right after angioplasty. In animal models, an efficient yet more economical way for new agent testing is a well-developed local intraluminal perfusion method. Candidate agents that have been tested through this method include small molecule drugs^13,14, cytokine or growth factors^15,16, gene manipulating agents (cDNA clones, siRNA, etc.)^17-20, and novel pharmaceutical formulations^21,22.

So far, the rat balloon injury model remains one of the most useful models for studying vascular diseases/disorders. It is the fundamental step from bench to bedside, usually as the first step moving from in vitro to in vivo, but it should not be the last one. The outcome of rat experiments needs to be deliberated and further characterized before translation into human clinical use, due to the difference in vascular beds and vessel anatomy as well as the intrinsic species differences between human and rat^23-26. Nevertheless, it is still an essential tool in translational medical research. While such research used to be limited by the lack of genetically modified rats, it has no longer been an issue since novel genomic approaches such as zinc-finger nucleases²⁷, TALENs²⁸ and CRISPR-Cas²⁹ have made knockout rats easily accessible.

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 anesthetic drugs intra-peritoneally (i.p.).
   1. Verify the adequacy of sedation by toe pinch in 5-10 min. Administer an additional small dose of drugs (ketamine 7 mg/kg and xylazine 0.6 mg/kg) if sedation is not complete.
   2. Make sure the needle is deep enough to delivery drugs intra-peritoneally because failure to deliver the entire drug solution into the peritoneal cavity will cause inadequate sedation.
      NOTE: Ulcers and hair loss in the skin at the injection site will be visible several days after surgery, due to inadvertent subcutaneous (s.c.) drug solution injection.
5. Inject 3 ml sterile saline solution subcutaneously (s.c.). Using a sterile cotton swab, apply a small amount of ophthalmic ointment to both eyes to prevent the corneas from drying out.
6. Prepare the heating equipment. Pre-warm heat pads by microwave or water bath.
7. Put the animal supinely on the surgical platform.
   1. Remove the hair in the ventral neck region. Apply the hair remover with a cotton swab, wait 30 sec and wipe off completely with gauze.
   2. Swab the neck with Povidone-Iodine scrub and 70% ethanol.
8. Put on personal protective equipment, including gown, hair cover, surgical mask, and glasses. Put on sterile surgical gloves at the end before handling the sterile surgical instruments and supplies.
9. Drape the rat with a sterile surgical sheet with only the neck region exposed.

2. Surgical Procedures

1. During the surgical procedure, check sedation depth of animal by toe pinch in every 15 min. If animal responds to toe pinch, add additional small dose (10% of the initial dose) of ketamine and xylazine.
2. Dissect left common carotid artery (CCA)
   1. Use a scalpel to make a straight longitudinal incision in the middle of neck. The approximate length of skin incision is 1.5-2 cm, for the purpose of isolating a 1.5-2 cm portion of artery. The length may vary depends on the purpose of study.
   2. Bluntly dissect the connective tissue from the skin. Keep the forceps tips up and make sure not to puncture the skin or the underlying tissue.
   3. Dissect muscle layers longitudinally along the left side of trachea.
   4. When opening up the muscle layer, visualize the left CCA with the vagus nerve closely attached. Bluntly dissect alongside the left carotid artery with extreme caution to separate the vagus nerve with minimal stretching.
   5. Dissect CCA distally till the bifurcation. Carefully dissect the bifurcation and two branches - internal carotid artery (ICA) and external carotid artery (ECA).
   6. Keep dissecting around the CCA, until approximately a portion of 1.5-2 cm of the artery is isolated from the surrounding tissues.
3. Balloon injury
   1. Permanently make a ligature on the ECA at approximately 5 mm away from bifurcation. Permanently ligate the occipital branch of ECA which is close to the bifurcation of ECA and ICA. Also permanently ligate other branches, if any, locating between the bifurcation and ECA ligature – for example, the superior thyroid branch. Suture that used for all ligatures is 4-0 black silk. Clip on the proximal end of CCA and the distal end of ICA.
      NOTE: Now, blood flow has been stopped either permanently through ligation (on ECA) or temporarily through clipping (on CCA and ICA). Luminal contents in the bifurcation area have been isolated from the systemic circulation.
   2. Make an arteriotomy incision on ECA by small-micro scissors. Ensure that the incision is close to the distal suture knot. Clean blood with saline and cotton swabs.
   3. Insert the uninflated 2F balloon catheter into the ECA lumen. Advance the balloon catheter proximally to the CCA lumen. Keep advancing the catheter proximally until its tip reaches where the clip stays.
   4. Connect the balloon inflation device to a female luer lock on a 3-way stopcock and connect the male luer lock of the 3-way stopcock to the balloon catheter.
   5. Slowly inflate the balloon with approximately 1.5 atm pressure, in order to distend the carotid artery to 1.5-fold of the diameter.
   6. Gently pull the balloon rotationally back to the bifurcation.
   7. Deflate the balloon and advance it back to the proximal end. Inflate it again and repeat the pulling-back procedure two more times.
   8. Withdraw the balloon catheter from the artery lumen.
4. Intraluminal administration of reagents (e.g., siRNA, drugs)
   NOTE: Here, the reagent used was a solution containing lentiviral particles encoding either shRNA targeting Stromal Interaction Molecule 1 (STIM1) or non-targeting shRNA control. STIM1 is a single transmembrane endoplasmic reticulum (ER) protein that is an ER Ca²⁺ sensor controlling the activation of plasma membrane Ca²⁺ channels and is up-regulated during vascular smooth muscle dedifferentiation into a proliferative migratory phenotype^12,30-33.
   1. Attach an intravascular over-the-needle catheter to a syringe (24 G, 1.6 cm). Aspirate 30 µl solution of the testing reagents. Insert the catheter into the same incision on ECA.
   2. Advance the catheter tip into the CCA and tie a piece of suture with a single knot on the ECA to fix the catheter and close the incision temporarily.
   3. Inject the testing reagent solution into the lumen of CCA. Keep the solution in the vessel lumen for 30 min. Keep the exposed tissue moist with saline and cover it with a piece of wet gauze.
   4. After incubation, aspirate the remaining solution. Loosen that single knot and withdraw the catheter.
   5. Tie the ECA with a piece of suture proximally to the arteriotomy incision. Make the knot as close as possible to the bifurcation.
5. Close up the wound.
   1. Since the balloon injury and catheter introduction may cause a leak or puncture of the blood vessel, remove the clip on the ICA and check if there is any leak. If bleeding is observed, apply a piece of gauze and apply gentle pressure to stop bleeding.
   2. Remove the other clip on CCA.
   3. Make sure there are no signs of bleeding, and then remove all clamps and other surgical instruments. Cut off excess sutures.
   4. Close wound using skin sutures (4-0 black silk). Swab on all sides of the closed wound with the Povidon-Iodine or other anti-septic/bactericide/virucide agent. Inject 3 ml of sterile saline s.c.

3. Postoperative Procedures

1. Keep rat on heat pad after surgery. Administer one dose of 0.05 mg/kg buprenorphine via intramuscular injection (i.m.) to the rat. During the recovery procedure, keep animals’ eyes and mouth moist. Monitor the animal until it is awake and ambulant.
2. Place the animal in a clean cage without any bedding until it is fully recovered. After recovery, the animal will be returned to the animal room and housed individually.

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