Procedure for Human Saphenous Veins Ex Vivo Perfusion and External Reinforcement

Summary

The mechanisms leading to the development of intimal hyperplasia (IH) and vein graft failure are still poorly understood. This study describes an ex vivo system to perfuse human veins under controlled flow and pressure. Furthermore the efficiency of external mesh reinforcement to limit the development of IH was evaluated.

Abstract

The mainstay of contemporary therapies for extensive occlusive arterial disease is venous bypass graft. However, its durability is threatened by intimal hyperplasia (IH) that eventually leads to vessel occlusion and graft failure. Mechanical forces, particularly low shear stress and high wall tension, are thought to initiate and to sustain these cellular and molecular changes, but their exact contribution remains to be unraveled. To selectively evaluate the role of pressure and shear stress on the biology of IH, an ex vivo perfusion system (EVPS) was created to perfuse segments of human saphenous veins under arterial regimen (high shear stress and high pressure). Further technical innovations allowed the simultaneous perfusion of two segments from the same vein, one reinforced with an external mesh. Veins were harvested using a no-touch technique and immediately transferred to the laboratory for assembly in the EVPS. One segment of the freshly isolated vein was not perfused (control, day 0). The two others segments were perfused for up to 7 days, one being completely sheltered with a 4 mm (diameter) external mesh. The pressure, flow velocity, and pulse rate were continuously monitored and adjusted to mimic the hemodynamic conditions prevailing in the femoral artery. Upon completion of the perfusion, veins were dismounted and used for histological and molecular analysis. Under ex vivo conditions, high pressure perfusion (arterial, mean = 100 mm Hg) is sufficient to generate IH and remodeling of human veins. These alterations are reduced in the presence of an external polyester mesh.

Introduction

Cardiovascular diseases are the leading cause of morbidity and mortality in Western countries¹. Despite advances made in endovascular treatments, bypass surgery remains the mainstay of contemporary therapies, thus over half a million vein grafts are performed annually in the United States. However, despite decades of research, 30-60% of lower extremity vein grafts fail within the first years due to intimal hyperplasia (IH)². Mechanical forces, particularly low shear stress (SS) and high wall tension, are pivotal in the initiation and development of this hyperplastic response^3,4. To address this issue, an ex vivo veins per....

Protocol

The Ethical Committee of the University of Lausanne approved the experiments, which are in accordance with the principles outlined in the Declaration of Helsinki of 1975, as revised in 1983 for the use of human tissues.

1. Human Great Saphenous Vein Harvest

1. Obtain surplus segments of non-varicose human saphenous veins from patients undergoing lower limb bypass surgery for ischemia. In the operating room, disinfect the entire leg with an iodine solution and drape the patient to expose the leg from the groin to the foot.
2. Make a median incision from the groin to the knee (leaving the interrupted skin portion).
3. H....

Results

The EVPS provides a valuable tool to independently assess the hemodynamic forces on human saphenous vein grafts remodeling and IH.

Figure 1 shows the perfusion chamber and the vein support. In Figures 1A and B, the vein support before (Figure 1A) and after (Figure 1B) assembly, respectively, is pictured. It is composed (from the top to the bottom) of 1 plain stainless steel tube measuring 9 cm, which serves as.......

Discussion

This study uncovers an ex vivo vein perfusion system (EVPS) to perform extensive hemodynamic studies in human veins. This system allows saphenous veins perfusion under defined hemodynamic parameters in the absence of aggravating inflammatory and growth factors released by circulating cells in vivo. Thus, it provides a better understanding of the underlying pathways involved in the control of IH in human veins grafts^5,11,12,15.

Reproducible and quantifiable hem.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
RPMI 1640 - Glutamax	Life Technologies	61870-010
Penicilline/Streptomycine/Fungizone	Bioconcept	4-02F00-H
Dextran from Leuconostoc spp. 500 g	Sigma-Aldrich	31390
Tampon PBS CHUV pH 7.1-7.3 1 L	Laboratorium und Grosse Apotheke Dr. G. Bichsel AG	100 0 324 00
Cryosectionning embedding medium - Tissue-Tek OCT Compound	Fisher Scientific	14-373-65
Silicon Tubing (Peroxide) L/S 16 (96400-16 ) - 7.5 m	Idex Health & Science GMBH	MF0037ST
Y-splitter	Idex Health & Science GMBH	Y-connector
35 mm Culture dish	Sigma-Aldrich	CLS430165-100EA
15 ml Falcon tube	BD Bioscence	352096
50 ml Falcon tube	BD Bioscence	352098
Gearing pump - Reglo-Z	Idex Health & Science GMBH	SM 895	App-Nr 03736-00194
Pump Head	Idex Health & Science GMBH	MI0008
Monitoring Kit TRANSPAC IV	icumedical	011-0J736-01
20 ml Syringes	B. Braun Medical SA	4612041-02
Etibon 3-0 FS-2	Ethicon- Johnson&Johnson	EH7346H
Mesh ProVena 6-8mm	B. Braun Medical SA	1105012-14
NaCl: Sodium chloride solution perfusion 0.9% (100 ml)	B. Braun Medical SA	534534
Masterflex L/S Standard Drive	Cole-Parmer Instrument Co	7521-10
Acquisition card	National Instruments	PCI-6024 E
Flowmeter module	Transonic Systems Inc.	TS410 and T402
Stopcock with 3-ways	BD Connexta Luerlock	394600
Millex Filter	Milian	SE2M229I04