A Rabbit Model of Durable Transgene Expression in Jugular Vein to Common Carotid Artery Interposition Grafts

Summary

This method describes the placement of interposition vein grafts in rabbits, the transduction of the grafts, and the achievement of durable transgene expression. This allows the investigation of physiological and pathological roles of transgenes and their protein products in grafted veins, and testing of gene therapies for vein graft disease.

Abstract

Vein graft bypass surgery is a common treatment for occlusive arterial disease; however, long-term success is limited by graft failure due to thrombosis, intimal hyperplasia, and atherosclerosis. The goal of this article is to demonstrate a method for placing bilateral venous interposition grafts in a rabbit, then transducing the grafts with a gene transfer vector that achieves durable transgene expression. The method allows the investigation of the biological roles of genes and their protein products in normal vein graft homeostasis. It also allows the testing of transgenes for the activities that could prevent vein graft failure, e.g., whether the expression of a transgene prevents the neointimal growth, reduces the vascular inflammation, or reduces atherosclerosis in rabbits fed with a high-fat diet. During an initial survival surgery, the segments of right and left external jugular vein are excised and placed bilaterally as reversed end-to-side common carotid artery interposition grafts. During a second survival surgery, performed 28 days later, each of the grafts is isolated from the circulation with vascular clips and the lumens are filled (via an arteriotomy) with a solution containing a helper-dependent adenoviral (HDAd) vector. After a 20-min incubation, the vector solution is aspirated, the arteriotomy is repaired, and flow is restored. The veins are harvested at time points dictated by individual experimental protocols. The 28-day delay between the graft placement and the transduction is necessary to ensure the adaptation of the vein graft to the arterial circulation. This adaptation avoids rapid loss of transgene expression that occurs in vein grafts transduced before or immediately after grafting. The method is unique in its ability to achieve durable, stable transgene expression in grafted veins. Compared to other large animal vein graft models, rabbits have advantages of low cost and easy handling. Compared to rodent vein graft models, rabbits have larger and easier-to-manipulate blood vessels that provide abundant tissue for analysis.

Introduction

Atherosclerosis is a chronic inflammatory disease in which lipid accumulation and inflammation in the blood vessel wall lead to narrowing of the vessel lumen, heart attacks, strokes, and loss of limbs^1,2. Percutaneous interventions (e.g., angioplasty and stenting) and medical therapy (e.g., statins and antiplatelet agents) are useful treatments for atherosclerosis; however, they are often ineffective in treating severe obstructive disease both in the coronary and peripheral circulations. Bypass grafting, using autogenous vein segments, remains a common procedure for treating patients with sev....

Protocol

All animal protocols and studies were approved by the University of Washington Office of Animal Welfare.

1. Pre-operation (for All Surgeries)

1. Anesthetize the rabbit with 30 mg/kg ketamine and 1.5 mg/kg xylazine by intramuscular injection (IM) in the paraspinous muscles.
   ​NOTE: Food and water are not restricted before surgery.
2. While waiting for sufficient depth of anesthesia, set up the tables in the preparation room and operating room (OR).
   1. Prepare the preparation room for shaving the rabbit's neck and the placement of the intravenous (IV) port in the ear (survival surgery only). Place the ....

Results

The technical proficiency of a new operator must be validated before the operator can use this method to generate experimental data. The first milestone that a new operator must achieve is consistent vein graft patency after both the initial vein-grafting surgery and the subsequent delayed-transduction surgery. Over 90% patency after each of the surgeries is desirable and achievable. The patency can be assessed non-invasively using transcutaneous ultrasound, which we typically perform on .......

Discussion

Critical steps in this protocol include the management of anesthesia, anticoagulation, surgical manipulation of the artery/grafted vein, and hemodynamic measurements of the grafted vein. Proper management of anesthesia is critical in this multiple survival surgery model that includes two relatively long operations (typically 3-3.5 h for bilateral vein grafting and 1.5-2.5 h for bilateral graft transduction). We have administered anesthesia both via a nosecone and by endotracheal intubation and found that the intubation i.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Disposables
3mL syringe with 24G needle	Becton Dickinson	309571	2x for vein graft surgery; 2x for gene transfer  surgery
1 mL syringe with 27G needle	Becton Dickinson	309623	2x for vein graft surgery, 5x for gene transfer surgery
19G needle	Becton Dickinson	305187	Gene transfer surgery
20 mL syringe, luer lock	Nipro Medical Corp	JD+20L
Catheters, 24Ga x 3/4”	Terumo Medical Products	SROX2419V
21G needle	Becton Dickinson	305165	Gene transfer surgery and for 20 mL syringe of saline
Gauze 4” x 4”	Dynarex	3242	~10-15 per surgery
3-0 silk suture	Covidien Ltd.	S-244
5-0 silk suture	Covidien Ltd.	S-182
7-0 polypropylene suture	CP Medical	8703P	Vein graft surgery
7-0 polypropylene suture	CP Medical	8648P	Gene transfer surgery
5-0 polyglycolic acid suture	CP Medical	421A
3-0 polyglycolic acid suture	CP Medical	398A
Alcohol swabs	Covidien Ltd.	6818	For the placement of I.V. line
Catheter plug	Vetoquinol	411498
Ketamine HCl, 100 mg/mL	Vedco Inc.	5098916106
Xylazine, 100 mg/mL	Akorn Inc.	4821
Lidocaine, 20 mg/mL	Pfizer	409427702
Marcaine 0.5%	Pfizer	409161050
Beuthanasia D-Special	Intervet Inc.	NDC 00061047305	Harvest surgery only
Buprenorphine HCl, 0.3 mg/mL	Patterson Veterinary	12496075705
Saline IV bag, 0.9% sodium chloride	Baxter	2B1309
Heparin  (5000 U/mL)	APP Pharmaceuticals	NDC 63323-047-10
Papaverine (3.5 mg/ml)	American Reagent Inc.	NDC 0517-4002-25	Diluted from 30mg/mL stock; Use 1 mL maximum
Fentanyl patch, 25 mcg/h	Apotex Corp.	NDC 60505-7006-2
Isoflurane	Multiple vendors	Catalog number not available
Viral vector			Gene transfer surgery only
Surgical Instruments
Metzenbaum needle holder 7" straight	Roboz	RS-7900
Operating scissors 6.5" straight blunt/blunt	Roboz	RS-6828
Needle holder /w suture scissors	Miltex	8-14-IMC
Castroviejo scissors	Roboz	RS-5658
Castroviejo needle holder, 5.75" straight with lock	Roboz	RS-6412
Stevens scissors 4.25" curved blunt/blunt	Roboz	RS-5943
Alm retractor 4" 4X4 5mm blunt prongs	Roboz	RS-6514	2x
Backhaus towel clamp 3.5"	Roboz		4x
Micro clip setting forceps 4.75"	Roboz	RS-6496
Micro vascular clips, 11 mm	Roboz
Surg-I-Loop	Scanlan International	1001-81M	5 cm length
Bonaccolto forceps, 4” (10 cm) long longitudinal serrations, cross serrated tip, 1.2mm tip width	Roboz	RS-5210
Dumont #3 forceps Inox tip size .17 × .10 mm	Roboz	RS-5042
Graefe forceps, 4” (10 cm) long serrated straight, 0.8 mm tip	Roboz	RS-5280
Halstead mosquito forceps,  5" straight, 1.3 mm tips	Roboz	RS-7110	2x
Halstead mosquito forceps,  5" curved, 1.3mm tips	Roboz	RS-7111
Jacobson mosquito forceps 5" curved extra delicate, 0.9 mm tips	Roboz	RS-7117
Kantrowitz forceps, 7.25" 90 degree delicate, 1.7 mm tips	Roboz	RS-7305
Tissue forceps 5", 1X2 teeth, 2 mm tip width	Roboz	RS-8162
Allis-Baby forceps, 12 cm, 4x5 teeth, 3 mm tip width	Fine Science Tools	11092-12	2x
Adson forceps, 12 cm, serrated, straight	Fine Science Tools	11006-12
Veterinary electrosurgery handpiece and electrode	MACAN Manufacturing	HPAC-1; R-F11
Surgical Suite Equipment
Circulating warm water blanket and pump	Multiple vendors	Catalog number not available
Bair hugger warming unit	3M	Model 505
IV infusion pump	Heska	Vet IV 2.2
Isoflurane vaporizer and scavenger	Multiple vendors	Catalog number not available
Veterinary multi-parameter monitor	Surgivet	Surgivet Advisor
Veterinary electrosurgery unit	MACAN Manufacturing	MV-9
Surgical microscope	D.F. Vasconcellos	M900	25X magnification for vein graft surgery; 16X magnification for gene transfer surgery