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Eur J Cardiothorac Surg 2000;18:400-403
© 2000 Elsevier Science NL

Endothelial function of human vena saphena magna prepared with different minimally invasive harvesting techniques

Alexander M. Fabricius, Andreas Oser, Anno Diegeler, Thomas Rauch, Friedrich W. Mohr

Abteilung für Herzchirurgie, Herzzentrum University of Leipzig, Russenstrasse 19, D-04289 Leipzig, Germany

Received 14 December 1999; received in revised form 12 July 2000; accepted 4 August 2000.

Corresponding author. Tel.: +49-341-865-1421; fax: +49-341-865-1485
e-mail: faba{at}medizin.uni-leipzig.de


   Abstract
Top
 Abstract
1. Introduction
 2. Patients
 3. Material and methods
 4. Results
 5. Comment
 References
 
Objectives: Minimally invasive saphenous harvesting techniques have been shown to reduce post-operative morbidity. However, when applying new techniques of vein harvesting, endothelial integrity should be preserved in order to guarantee graft quality. We investigated the impact of two different minimally invasive saphenous vein harvesting techniques on endothelial function compared with the traditional ‘open’ technique by inducing endothelium-dependent in vitro relaxation. Methods: Two different minimally invasive techniques for harvesting the greater saphenous vein were used in 66 patients, either using a video-assisted dissector (ETHI, n=33) or a light coupled retractor (AUTS, n=33); other patients were treated conventionally (CONV, n=30). The physiological response was tested in vitro using an organ chamber on vein segments exposed to acetylcholine after precontraction with potassium. Results: In both minimally invasive groups, two patients each had to be converted to the traditional open technique and dropped out of the study. The endothelial function of the other veins harvested by either of the minimally invasive techniques showed no significant difference compared with veins harvested conventionally. Reactivity was measured as the percentage relaxation of pharmacological contraction and was significant in all groups (P<0.05); ETHI, 49.1±4.2%; AUTS, 48.8±5.1%; and CONV, 51.7±6.0%. The responder/non-responder ratio was similar in all groups: ETHI (two drop-outs), 28/3 (90.3%); AUTS (two drop-outs), 28/3 (90.3%); and CONV, 27/3 (90.0%). Conclusion: Veins harvested by minimally invasive techniques have not shown an impaired reactivity of the endothelium compared with the conventional technique.

Key Words: Vena saphena magna • Minimally invasive technique • Endothelial function


   1. Introduction
Top
 Abstract
 1. Introduction
2. Patients
 3. Material and methods
 4. Results
 5. Comment
 References
 
The functional integrity of the endothelium in venous conduits used for coronary bypass may be of importance for early or late graft occlusion [1,2]. Surgical techniques used to harvest venous conduits are important in terms of the potential to damage the vessel wall [3], and influence the early patency of coronary bypass vein grafts, as shown in angiographic and ultrastructural findings [4].

Various minimally invasive techniques of saphenous vein harvesting have been shown to reduce post-operative morbidity [5,6]. This in vitro study was undertaken in order to investigate the contractile responses of the autologous undistended saphenous vein comparing two minimally invasive techniques and the traditional ‘open’ technique by exposing segments of harvested veins to constricting and dilating agents.


   2. Patients
Top
 Abstract
 1. Introduction
 2. Patients
3. Material and methods
 4. Results
 5. Comment
 References
 
Ninety-six patients were randomly assigned to three groups using different techniques of vein harvesting; one using a video-assisted dissector (EthiconTM; ETHI, n=33) [5], one using a light coupled retractor (AutoSutureTM; AUTS, n=33) [6], and one using the conventional ‘open’ technique of vein harvesting (CONV, n=30) which served as a control group. All procedures were performed by the same surgeon. Pre-operative exclusion criteria included peripheral vascular disease, visible varicosities, and pre- or intra-operative catecholamine therapy. All three groups were comparable in terms of patient characteristics; gender, age, obesity and diabetes mellitus. Randomization was done in an alternating manner. The analysis was performed with the intention to treat methodology. Patients converted to the open technique dropped out. Venous samples were taken from either the proximal, mid or distal end of the harvested and undistended vein.


   3. Material and methods
Top
 Abstract
 1. Introduction
 2. Patients
 3. Material and methods
4. Results
 5. Comment
 References
 
3.1. General
During harvesting, no infusion was used. Having prepared the veins conventionally or with one of the minimally invasive techniques as previously described [5,6], a 2 cm non-distended segment was taken and immediately stored in an aerated, Krebs–Henseleit solution at 4°C, taken to the lab within 5 min, carefully dissected out of its surrounding tissue, and three equal rings of approximately 3 mm width were prepared. The composition of the Krebs–Henseleit solution was as follows: 144 mmol/l Na+; 5.9 mmol/l K+; 2.5 mmol/l Ca+; 1.2 mmol/l Mg2+; 128.7 mmol/l Cl-; 25 mmol/l HCO3-; 1.2 mmol/l SO4-; 1.2 mmol/l H2PO4-; and 11 mmol/l glucose.

3.2. Organ bath
The vein rings were mounted between two hooks immersed in a 5 ml organ bath containing Krebs–Henseleit solution, as previously described [7]. The solution was maintained at a temperature of 37°C and gassed with 95% oxygen and 5% carbon dioxide. The upper hook was controlled by a micrometer connected to a strain gauge linked to a recorder and computer (Technical and Scientific Equipment, Bad Homburg), thereby allowing the measurement of wall force and length (internal circumference of the venous ring). The venous rings were exposed to a defined resting tension, and stretched thereafter in steps to a standard point on the vein's individual length–passive wall tension curve. The internal radius (R) of each venous ring could be determined by iterative fitting of pressure-dependent isobars intersecting the exponential length–tension curve by a computer software program (Technical and Scientific Equipment, Bad Homburg). Depending on the individual length–tension curve of each vein, the stretching force was stopped when a transmural pressure of 60 mmHg was reached, thereby defining the internal radius (R100). The transmural pressure was set to 60 mmHg instead of 20 or 50 mmHg, as pilot studies have shown that the reactivity was clearer at this setting. The rings were then released to 90% of their internal circumference. The isometric force at this setting has been termed the ‘passive’ or ‘resting force’ in the absence of any constrictor tone. After equilibrating the three rings for 1 h, 2x10-4 M potassium chloride (KCl) was added to each organ bath and the contracting wall force was read by the recorder. When a steady state was reached, 4x10-6 M acetylcholine was added to induce relaxation in two chambers and normal saline solution was added as a reference in a third chamber, thus running three organ bath arrangements concurrently. Relaxation was expressed as the percentage relaxation of the postcontraction wallforce induced by KCl and measured for 30 min. The concentrations of KCl and acetylcholine used were tested in pilot experiments and were in accordance with the literature. A representative contraction/relaxation-force time chart is presented in Fig. 1 .



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  		Fig. 1. Typical graph showing endothelium-dependent relaxation induced by acetylcholine (4x10-6 M) treatment of an isolated vein ring after precontraction with 2x10-4 potassium in the organ chamber. The dip in the middle of the recording results from changing the bathing medium.

 
3.3. Data analysis
Cumulative dose response curves were omitted because we wished to only evaluate simple reactivity. The term ‘reactivity’ throughout the text describes the response in terms of the maximum relaxation (or contraction) to the pharmacological vasodilator (or vasoconstrictor) substance, expressed as a percentage of the precontracted (or predilated) force regardless of any location on the response curve.

All statistical data presented in Figs. 1 and 2 and Table 1 are means±SD. The Student's two-tailed unpaired t-test was used to test statistical significance between rings. The statistical significance of possible differences in quantifiable variables was done by analysis of variance (ANOVA). The Bonferroni adjustment procedure was applied for multiple comparisons of mean values. Statistical significance was defined at P values of <0.05. Statistical calculations were performed using SPSS (Release 10.0, SPSS, Inc.).



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  		Fig. 2. Endothelium-dependent relaxation of isolated precontracted vein rings after application of acetylcholine (4x10-6 M).

 

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  		Table 1. Summary of vessel parameters exposed to KCl and acetylcholinea,b

 

   4. Results
Top
 Abstract
 1. Introduction
 2. Patients
 3. Material and methods
 4. Results
5. Comment
 References
 
Two patients each in both minimally invasive groups had to be converted to the traditional open technique due to strong adhesions tethering the vein to the skin, thus excluding these veins for this study and leaving 31 study veins each in the minimally invasive groups and 30 study veins in the conventional group.

4.1. Resting vessel parameters
The internal diameters of the prepared vein rings were similar in all groups: ETHI, 912±18 (n=62); AUTS, 923±21 (n=62); and CONV, 918±20 µm (n=60).

4.2. Contraction
Reactivity to 2x10-4 M KCl was seen in all prepared vein rings: ETHI, 18.3±0.7 (n=62); AUTS 17.2±0.6 (n=62); and CONV, 16.6±0.6 mN (n=60).

4.3. Relaxation
The responder/non-responder ratio of veins was similar in all groups since both vein rings of the same three vessels in every group did not show any reactivity at all: ETHI (two drop-outs), 28/3 veins (90.3%); AUTS (two drop-outs), 28/3 veins (90.3%); and CONV, 27/3 veins (90.0%). The application of 4x10-6 M acetylcholine relaxed the vein rings of CONV (n=60) to a maximum of 8.6±0.6 mN (51.7±3.6%), compared with the precontracted vein rings, followed by ETHI with 9.0±0.8 (49.1±4.2%; n=61) and AUTS with 8.4±0.9 mN (48.8±5.1%; n=61).

The application of saline solution resulted in the relaxation of rings ranging from 1.02±0.1 (5.6±0.02%; n=31) in ETHI and 0.93±0.1 (5.6±0.02%; n=31) in CONV to 0.9±0.1 mN (5.1±0.02%; n=31) in AUTS. We accounted all effects to a rinsing effect.


   5. Comment
Top
 Abstract
 1. Introduction
 2. Patients
 3. Material and methods
 4. Results
 5. Comment
References
 
A semi-closed technique for the subcutaneous removal of the saphenous vein was first described by Mayo in 1906 [8], and its use for harvesting the saphenous vein has been retrospectively analyzed in two series [9,10]. Meanwhile, various new minimally invasive techniques using commercially available instruments have been introduced and their clinical applicability has been shown. However, the potential increase in handling of the saphenous vein while employing new dissecting techniques may be detrimental to the conduit. Graft quality and the subsequent occlusion of the coronary artery bypass graft are dependent on the structural and functional integrity of the conduits used to bypass the desired vessels. Impairment of biological properties, such as the endothelial layer, has been shown to cause myointimal proliferation [11], affecting short- and long-term graft performance [1,2]. Most of the factors impairing the endothelium occur during [12,13] or after surgical preparation, when distending [14,15] or stretching [16] the graft. Surgical techniques used to harvest venous conduits are to preserve, thereby guaranteeing graft quality. Previous studies on porcine veins [17] and human saphenous veins [18] evaluated the morphological integrity by histology and scanning electron microscopy, and have shown the endothelial layer to be preserved. Biological endothelial integrity is required for NO production in the saphenous veins and internal mammary artery [19], and inhibition of platelet deposition[20]. Few studies have assessed vascular reactivity and surgical techniques [1,21], or compared different techniques [22], especially minimally invasive techniques [17,23,24].

In our study, we assessed the biological properties of harvested veins by exposure to a non-specific vasoconstrictor, such as KCl, and an endothelial specific vasodilator, such as acetylcholine [11,25]. There was no statistically significant difference between the vascular reactivity of the two minimally invasive techniques and the conventional technique (Fig. 2).

However, there are limitations to the experimental set-up that need to be discussed. Noradrenaline for contraction is probably the better agent for precontraction compared with KCl, because it does not impair the release of nitric oxide due to cell depolarization. Hence, more reliable data could have been obtained. Relaxation was induced by acetylcholine alone; for comparison, an endothelium-independent vasodilator, such as sodium nitroprusside, would have been helpful, besides bradykinin or the nitric oxide donor, SIN-1, as published before [26]. A cumulative fashion of application could have resulted in different dose–response curves that may have revealed subtle changes in endothelial function.

Within the limitations of our experimental set-up, the results were surprising to us, as the mechanical force applied to veins prepared minimally invasively seemed to be higher than in the conventional group. Having seen the early findings of vein performance with KCl and acetylcholine, we decided to perform additional tests with 4x10-7 M noradrenaline bitartrate for smooth muscle function, and again, veins of all groups showed a similar contraction. On the other hand, the maximum wall force applied by distending veins, regardless of the preparation technique, showed neither contraction nor relaxation. These results were not assessed statistically because we had already started the study and were limited by the preparation kits, but we found these results to be noteworthy, because they may underline the fact that veins seem to be more vulnerable to transverse force than longitudinal force as applied to the vein when harvesting with minimal invasiveness.

Histological studies of the veins taken with different minimally invasive techniques should be performed timewise to assess the endothelial integrity, since endothelial denudation leads to intimal medial repair with neointimal thickening [27]. For long-term quality control, post-operative assessment of graft patency by angiography may give further insight into possible differences between conventional and minimally invasive harvesting techniques. A larger cohort of patient veins will have be investigated in the future to evaluate possible differences of harvesting techniques.


   References
Top
 Abstract
 1. Introduction
 2. Patients
 3. Material and methods
 4. Results
 5. Comment
 References
 

  1. Dhein S., Reiss N., Gerwin R., Borowski A., Korb H., Klaus W., de Vivie E.R. Endothelial function and contractility of human vena saphena magna prepared for aortocoronary bypass grafting. Thorac Cardiovasc Surg 1991;39:66-69.[Medline]
  2. He G.W., Rosenfeldt F.L., Angus J.A. Pharmacological relaxation of the saphenous vein during harvesting for coronary artery bypass grafting. Ann Thorac Cardiovasc Surg 1989;97:259-266.
  3. Kennedy J.H., Lever M.J., Addis B.J., Paneth M. Changes in vein interstitium following distension for aortocoronary bypass. J Cardiovasc Surg 1989;30:992-995.[Medline]
  4. Catinella F.P., Cunningham J.N., Jr, Srungaram R.K., Baumann F.G., Nathan I.M., Glassmann E.A., Knopp E.A., Spencer F.C. The factors influencing early patency of coronary bypass vein graft: correlation of angiographic and ultrastructural findings. J Thorac Cardiovasc Surg 1982;83:686-700.[Abstract]
  5. Cable D.G., Dearani J.A. Endoscopic saphenous vein harvesting: minimally invasive video-assisted saphenectomy. Ann Thorac Surg 1997;64:1183-1185.[Abstract/Free Full Text]
  6. Tevaearai H.T., Mueller X.M., von Segesser L.K. Minimally invasive harvest of the saphenous vein for coronary artery bypass grafting. Ann Thorac Surg 1997;63(Suppl 6):S119-S121.
  7. He G.W., Angus J.A., Rosenfeldt F.L. Reactivity of the canine isolated internal mammary artery, saphenous vein, and coronary artery to constrictor and dilator substances: relevance to coronary bypass graft surgery. J Cardiovasc Pharmacol 1988;12:12-22.[Medline]
  8. Mayo C.H. Treatment of varicose veins. Surg Gynecol Obstet 1906;2:385-388.
  9. Dimitri W.R., West I.E., Williams B.T. A quick and atraumatic method of autologous vein harvesting using the subcutaneous extraluminal dissector. J Cardiovasc Surg 1987;39:66-69.
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  12. Angelini G.D., Christie M.I., Bryan A.J., Lewis M.J. Surgical preparation impairs release of endothelium-derived relaxing factor from human saphenous vein. Ann Thorac Surg 1989;48:417-420.[Abstract]
  13. Gundry S.R., Jones M., Ishihara T., Ferrans V.J. Intraoperative trauma to saphenous veins: scanning electron microscopic comparison of preparation techniques. Ann Thorac Surg 1980;30:40-47.[Medline]
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  15. Hasse J., Graedel E., Hofer H., Guggenheim R., Amsler B., Mihatsch M.J. Morphologic studies in saphenous vein grafts for aorto-coronary bypass surgery. Part II: influence of pressure-limited graft dilatation. Thorac Cardiovasc Surg 1981;29:38-40.[Medline]
  16. Bush H.L., Jr, Jakubowski J.A., Curl G.R., Deykin D., Nabseth D.C. The natural history of endothelial structure and function in arterialized vein grafts. J Vasc Surg 1986;3:204-215.[Medline]
  17. Cable D.G., Dearani J.A., Pfeifer E.A., Daly R.C., Schaff H.V. Minimally invasive saphenous vein harvesting: endothelial integrity and early clinical results. Ann Thorac Surg 1998;66:139-143.[Abstract/Free Full Text]
  18. Fabricius A.M., Diegeler A., Doll N., Weidenbach H., Mohr F.W. Two different minimally invasive techniques for greater saphenous vein harvesting: morphology and postoperative outcome. Ann Thorac Surg 2000;70:473-478.[Abstract/Free Full Text]
  19. Lüscher T.F., Diederich D., Siebenmann R., Lehmann K., Stulz P., von Segesser L., Zhihong Y., Turina M., Grädel E., Weber E., Bühler F.R. Difference between endothelium-dependent relaxation in arterial and in venous coronary bypass grafts. N Engl J Med 1988;319:462-467.[Abstract]
  20. Radomski M.W., Palmer R.M., Moncada S. Modulation of platelet aggregation by an L-arginine–nitric oxide pathway. Trends Pharmacol Sci 1991;12:87-88.[Medline]
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  24. O'Regan D.J., Borland J.A.A., Chester A.H., Pennell D.J., Yacoub M., Pepper J.R. Assessment of human long saphenous vein function with minimally invasive harvesting with the Mayo stripper. Eur J Cardio-thorac Surg 1997;12:428-435.[Abstract]
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