Name: procedure for human saphenous veins ex vivo perfusion and external reinforcement
Uploaded: 2014-10-01T15:00:00
Description: Watch this Scientific Journal Video about Procedure for Human Saphenous Veins Ex Vivo Perfusion and External Reinforcement at JoVE.com

This work was supported by grants from the SNF [31003A-138528], the Octav and the Marcella Botnar Foundation, the Novartis Foundation and the Emma Muschamp Foundation. We thank Martine Lambelet, and&#160;Jean-Christophe Stehle for their excellent technical assistance.

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
<ol>
	<li>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 expo...

<ol>
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This study uncovers an ex&#160;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 grafts5,11,12,15.
Reproducible and quantifiable hem...

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 response3,4. To address this issue, an ex&#160;vivo veins perfusion system (EVPS) was generated to study, under strictly controlled hemodynamic conditions (pressure and shear stress), the behavior of human saphenous veins. In this study, following insertion into the arterial-like circulation, high pressure (mean = 100 mm Hg) was sufficient to stimulate proliferation and migration of smooth muscle cells into the intimal layer (IH)5.
Mammalian studies have suggested the use of external reinforcement as an efficient method to support the &#8220;arterialized vein&#8221; and counteract the acute hemodynamic changes the vein faces once implanted into an arterial milieu. The mesh prevented over-distension, increased shear stress, and reduced wall tension and consequently IH6-10. However, the underlying mechanisms and its applicability to human veins in improving bypass patency have not been fully characterized. Our EVPS was used to compare, in condition mimicking the alterations a vein faces once inserted into an arterial regimen (high shear stress and pressure), the behavior of human saphenous veins in the absence and presence of an external macroporous polyester tubular mesh. By preventing pathological remodeling and IH, the mesh provided evidence of its potential clinical efficiency11.
This study 1) introduces a model of ex&#160;vivo human saphenous veins perfusion under controlled pressure and shear stress 2) demonstrates that external macro-porous polyester mesh reduces IH and provides crucial information for its potential clinical application.

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			<td height="21" style="height:21px;width:453px;">Name</td>
			<td style="width:371px;">Company</td>
			<td style="width:145px;">Catalog Number</td>
			<td style="width:144px;">Comments</td>
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			<td height="21" style="height:21px;">RPMI 1640 - Glutamax</td>
			<td>Life Technologies</td>
			<td>61870-010</td>
			<td></td>
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			<td height="21" style="height:21px;"><a name="RANGE!A3">Penicilline/Streptomycine/Fungizone</a></td>
			<td>Bioconcept</td>
			<td>4-02F00-H</td>
			<td></td>
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			<td height="21" style="height:21px;">Dextran from Leuconostoc spp. 500 gr.</td>
			<td>Sigma-Aldrich</td>
			<td>31390</td>
			<td></td>
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			<td height="21" style="height:21px;">Tampon PBS CHUV pH 7.1-7.3 1 lt.</td>
			<td>Laboratorium und Grosse Apotheke Dr. G. Bichsel AG</td>
			<td>100 0 324 00</td>
			<td></td>
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			<td height="21" style="height:21px;">Cryosectionning embedding medium - Tissue-Tek OCT Compound</td>
			<td>Fisher Scientific</td>
			<td>14-373-65</td>
			<td></td>
		</tr>
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			<td height="21" style="height:21px;">Silicon Tubing (Peroxide) L/S 16 (96400-16 ) - 7.5m</td>
			<td>Idex Health &#38; Science GMBH</td>
			<td>MF0037ST</td>
			<td></td>
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			<td height="21" style="height:21px;">Y-splitter&#160;</td>
			<td>Idex Health &#38; Science GMBH</td>
			<td>Y-connector</td>
			<td></td>
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			<td height="21" style="height:21px;">35 mm Culture dish</td>
			<td>Sigma-Aldrich</td>
			<td>CLS430165-100EA</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">15 ml Falcon tube</td>
			<td>BD Bioscence</td>
			<td>352096</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">50 ml Falcon tube</td>
			<td>BD Bioscence</td>
			<td>352098</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gearing pump - Reglo-Z</td>
			<td>Idex Health &#38; Science GMBH</td>
			<td>SM 895&#160;</td>
			<td>&#160;App-Nr 03736-00194</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pump Head</td>
			<td>Idex Health &#38; Science GMBH</td>
			<td>MI0008&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Monitoring Kit TRANSPAC IV</td>
			<td>icumedical</td>
			<td>011-0J736-01</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">20 mL Syringes</td>
			<td>B. Braun Medical SA</td>
			<td>4612041-02</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Etibon 3-0 FS-2</td>
			<td>Ethicon- Johnson&#38;Johnson</td>
			<td>EH7346H</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mesh ProVena 6-8mm</td>
			<td>B. Braun Medical SA</td>
			<td>1105012-14</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NaCl: Sodium Chlorure Solution perfusion 0.9% (100 ml)</td>
			<td>B. Braun Medical SA</td>
			<td>534534</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Masterflex L/S Standard Drive</td>
			<td>Cole-Parmer Instrument Co</td>
			<td>7521-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Acquisition card</td>
			<td>National Instruments</td>
			<td>PCI-6024 E</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Flowmeter module</td>
			<td>Transonic Systems Inc.</td>
			<td>TS410 and T402</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Stopcock with 3-ways</td>
			<td>BD Connexta Luerlock</td>
			<td>394600</td>
			<td></td>
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			<td height="21" style="height:21px;">Millex Filter</td>
			<td>Milian</td>
			<td>SE2M229I04</td>
			<td></td>
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procedure for human saphenous veins ex vivo perfusion and external reinforcement

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&#160;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&#160;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.

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...

Watch this Scientific Journal Video about Procedure for Human Saphenous Veins Ex Vivo Perfusion and External Reinforcement at JoVE.com

Procedure for Human Saphenous Veins Ex Vivo Perfusion and External Reinforcement

The mechanisms leading to the development of intimal hyperplasia (IH) and vein graft failure are still poorly understood. This study describes an ex&#160;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.

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