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Logo of thijTexas Heart Institute JournalSee also Cardiovascular Diseases Journal in PMCSubscribeSubmissionsTHI Journal Website
 
Tex Heart Inst J. 2007; 34(3): 290–295.
PMCID: PMC1995050

Immunohistochemical Comparison of Traditional and Modified Harvesting of the Left Internal Mammary Artery

Abstract

The left internal mammary artery is the conduit of choice for coronary artery bypass grafting. In the traditional (“clipped-artery”) harvesting technique, this artery is prepared as a pedicle; the distal part is clipped, cut, and covered with a papaverine-soaked cloth until anastomosis is performed. In modified (“nonclipped-artery”) harvesting, the prepared artery is kept in situ and left connected to the systemic circulation until anastomosis. Better outcomes from use of the nonclip technique have been reported. In order to determine comparative endothelial integrity and endothelial nitric oxide synthase activity, we performed an immunohistochemical study of arterial graft segments that were procured by each technique.

This cross-sectional study involved 40 patients who underwent elective coronary artery bypass grafting. The patients were randomized into 2 groups of 20. One group underwent traditional clipped-artery harvesting; the other group, modified nonclipped-artery harvesting. By immunohistochemical methods, we examined redundant segments taken from bifurcation levels of the arteries.

The tunica media was thinner in the clipped arterial segments, a phenomenon that we attribute to high luminal pressure. Endothelial nitric oxide synthase immunostaining was absent in regions of denudation in the luminal endothelia of the clipped arteries; in contrast, pronounced immunostaining occurred in the endothelia of the nonclipped segments.

We found that traditional harvesting disrupted the integrity of the luminal endothelia of the clipped arteries. In addition, the traditional procedure decreased nitric oxide production, as was revealed by immunostaining.

Key words: Coronary artery bypass/methods, coronary disease/surgery, endothelium, vascular/physiology, graft occlusion, vascular, graft survival, immunohistochemistry, internal mammary-coronary artery anastomosis/adverse effects/methods, mammary arteries/transplantation, myocardial revascularization/methods, nitric oxide synthase/analysis/metabolism/type III, tunica media/enzymology/pathology

Since the late 1970s, the left internal mammary artery (LIMA) has routinely been used as the conduit of choice for coronary artery bypass grafting (CABG). Performing a LIMA bypass in operations on the left anterior descending branch of the coronary arteries is the gold standard today.1,2 Postoperative long-term patient survival and graft patency rates after CABG have improved as a result of the use of the LIMA rather than the saphenous vein.1–6 Intimal hyperplasia in venous grafts starts to develop immediately after CABG, and, in 30% of peripheral venous grafts, stenotic lesions begin to appear after 1 year.7 In comparative studies, the LIMA better resisted the development of atherosclerosis, intimal hyperplasia, and medial calcification than did venous grafts.1,2,8,9 Accordingly, use of the LIMA decreases risks of CABG patients' experiencing postoperative late-term myocardial infarction, cardiac events, and the need for reoperation, thus improving long-term survival.1,2

Nitric oxide (NO) prevents smooth-muscle mitogenicity that results in intimal thickening.10 Endogenous NO is synthesized by the endothelial NO synthase enzyme (e-NOS), which catalyzes the production of l-citrulline from l-arginine.11 Type III e-NOS is evident in the tunica intima and tunica media of all types of arteries, particularly in the LIMA and the radial artery. The endothelial NO synthase enzyme is most prominently expressed in the intima of the LIMA.12

In the harvesting method that traditionally precedes CABG, the LIMA is prepared as a pedicle; the distal part is clipped, cut, and covered with a papaverine-soaked cloth until anastomosis is performed. The prepared arterial segment is dilated by blood pressure, but its endothelial lining is damaged by the pressure between the time of harvesting and anastomosis. Alternately, in order to reduce the endothelial and medial damage caused by blood pressure, the LIMA can be prepared and kept in situ, connected to the systemic circulation until the time of anastomosis. According to reports,13,14 better overall function results after application of this modified, “nonclip” technique.

Levels of NO are elevated in LIMA grafts in general, and specifically in LIMA grafts procured by the nonclip technique; accordingly, graft function is better when the nonclip technique is used.13,14 Taking this into account, we harvested LIMA grafts by each technique and used immunohistochemical methods to determine comparative endothelial integrity and e-NOS activity. Our search of the medical literature indicates that ours is the 1st study in which the 2 techniques have been compared immunohistochemically.

Patients and Methods

We conducted a cross-sectional study of 40 patients who underwent elective CABG. Permission was obtained from our local ethics committee, and all patients gave their written informed consent.

The study population comprised consecutive patients who had been diagnosed with coronary artery disease and who would benefit from elective CABG. Forty patients were randomized into 2 groups of 20. In 1 group, the LIMA was harvested by the traditional clipped-artery technique; the other group underwent modified (nonclipped-artery) harvesting. We then immunohistochemically examined redundant segments taken from bifurcation levels of the arteries.

Preparation of the Arteries

Clipped Arteries. The LIMA was prepared as a pedicled graft. After systemic heparin administration, the artery was clipped, wrapped in a papaverine-soaked cloth, and stored under the manubrium sterni until anastomosis. After the institution of extracorporeal circulation and cardioplegic arrest of the heart, peripheral vein anastomoses were performed. A redundant 1-cm-long arterial segment proximal to the clip was cut off and preserved in formalin for laboratory examination.13

Nonclipped Arteries. Nonclipped arteries were prepared as pedicles and were left perfused, connected to blood flow in situ at the distal end, until anastomosis. A redundant 1-cm-long arterial segment proximal to the clip was cut off and preserved in formalin for laboratory examination.13

Histopathologic Procedure. All harvested arterial segments were immersed in 10% neutral formalin solution and embedded in paraffin. Sections were cut with a cryostat at 6-μm thickness and were prepared for histochemical and immunohistochemical studies. These sections were evaluated for luminal endothelial integrity in the LIMA and in the vaso vasorum.

Immunohistochemistry. For the immunohistochemical studies, immunostaining was performed by use of the streptavidin-biotin-peroxidase complex technique. The sections in paraffin were collected on slides; the paraffin was removed, and the sections were rehydrated. Endogenous peroxidase activity was blocked by use of 3% hydrogen peroxide. The sections were incubated with primary antisera, including RB-1711-R7 endothelial nitric oxide synthase AB-1 rabbit polyclonal antibody (Lab Vision Corp., part of Thermo Fisher Scientific Inc.; Fremont, Calif). After washing in phosphate-buffered saline, the tissues were incubated with a biotin-conjugated secondary antibody and then incubated by use of the streptavidin-biotin system for 30 min at room temperature. The reactions became visible after immersion of the specimens in diaminobenzidine tetra-hydrochloride. The sections were counterstained with hematoxylin and eosin (H&E) stain, then rinsed and mounted.

The expression intensity of e-NOS was detected immunohistochemically. The intensity of the immuno-staining was graded semiquantitatively on a scale ranging from 0 (the absence of staining) to 4 (diffuse and intense staining).

Statistical Analysis

Statistical analysis was carried out by use of SPSS for Windows v. 11.0 (SPSS Inc.; Chicago, Ill). Variables were presented as mean ± SD. The normal distribution of variances among groups for continuous variables was evaluated by Levene's test. The χ2 test was used to compare dichotomous variables, the Student t test for continuous variables, and the Mann-Whitney U test for the difference between groups in regard to e-NOS staining. P values ≤0.05 were considered statistically significant.

Results

The patient groups were compared for mean age, body mass index (BMI), and time until anastomosis; no significant differences were found (Table I). Risk factors and preoperative medication use were not significantly different between the groups (Table II). The e-NOS immunostaining intensities in the luminal endothelia of the LIMA segments are shown in Table III.

Table thumbnail
TABLE I. Comparison of Mean Age, Body Mass Index, and Time until Anastomosis
Table thumbnail
TABLE II. Comparison of the Groups Regarding Risk Factors and Medication Use
Table thumbnail
TABLE III. Intensity of Immunostaining in the Luminal Endothelium of LIMA Segments in Determination of the Presence of e-NOS

The tunica media was thinner in the LIMA segments harvested by the clipped-artery technique; we attribute this to high luminal pressure (Fig. 1). The e-NOS immunostaining was absent in regions of denudation in the luminal endothelia of the clipped arteries, but it was pronounced in the same areas of the nonclipped arteries (Fig. 2).

figure 5FF1
Fig. 1 Hematoxylin & eosin-stained sections of arteries harvested by clip and nonclip techniques. A) The tunica media is thinner in the segments prepared by the clip technique, apparently due to high luminal pressure (orig. ×200). B) Tunica ...
figure 5FF2
Fig. 2 Immunohistochemical endothelial nitric oxide synthase (e-NOS) sections of arteries harvested by the clip and nonclip techniques (biotin-streptavidin stain, orig. ×400). A) In the clip technique, e-NOS immunostaining is absent in regions ...

Although no immunostaining was noted in the endothelial linings of the vaso vasorum in the clipped-artery group (Fig. 3A, arrow), dense immunostaining of those parts was seen in the nonclipped arteries (Fig. 3B, arrow).

figure 5FF3
Fig. 3 Immunohistochemical endothelial nitric oxide synthase (e-NOS) sections of arteries harvested by the clip and nonclip techniques. A) In the clip technique, weak adventitial immunostaining of e-NOS is revealed in the vaso vasorum (arrow) (biotin-streptavidin ...

Discussion

The LIMA is accepted as an ideal conduit for myocardial revascularization.12,15 As shown in biological, morphologic, and angiographic studies, this artery is protected from atherosclerosis even in patients who have advanced atherosclerosis.1,16,17

Nitric oxide plays an important part in vasodilation and resistance to atherosclerosis in the human cardiovascular system.11,18–21 Studies have shown that basal and stimulated NO secretion is significantly higher in the LIMA than in other vessels.12,22 This characteristic may account for the resistance to atherosclerosis and for the long-term patency rates of LIMA grafts.10,23,24 Three isoenzymes of nitric oxide synthase (NOS) are present in mammalian cells.12 It is generally accepted that NOS type III (e-NOS) is located in the vascular endothelium.25,26

In a previous study that used scanning electron microscopy,13 endothelial cell derangements indicated endothelial cell dysfunction in LIMA grafts that were harvested by the traditional clipped-artery technique. Also in this research effort, endothelial dysfunction was confirmed by comparing P-selectin and thrombomodulin levels in the blood. As reported in another study,14 stasis and arterial wall tension obstacles were overcome by the nonclipped-artery modification of the LIMA graft-preparation technique, which led to highly protected endothelial cell integrity and function. It can therefore be said that histochemical and morphologic differences occur in LIMA grafts that are prepared by means of the respective techniques.13

In our study, we attribute the decrease in tunica media thickness seen on H&E staining (Fig. 1) to the effect of luminal pressure after the arteries were clipped, as was mentioned in earlier studies.13,14 Our immunohisto-chemical findings support the conclusion that the increase in wall tension and luminal stasis resulting from the clipped-artery technique causes the disruption of luminal endothelial integrity and therefore disrupts endothelial function. Our e-NOS staining showed that e-NOS in the nonclipped LIMA segments was significantly higher than in the clipped LIMA segments.

It has been stated27–30 that loss of the vaso vasorum can cause ischemia in the tunica media and that concomitant loss of the draining veins can induce stasis and edema in the vascular wall. Arteries as large as 350 to 600 μm in diameter were found to be nourished by luminal diffusion.31 Sasajima and colleagues32 showed that the largest segment of the LIMA was approximately 152 μm in diameter; accordingly, the LIMA can be nourished by luminal diffusion even after total disruption of the vaso vasorum. In our study, we observed dense e-NOS immunostaining in the adventitial vaso vasorum of the nonclipped group, compared with no staining in the clipped group. The significance of this finding warrants further evaluation by electron-microscopic and biochemical studies that can depict the effects of vaso vasorum damage on vessel wall integrity.

We conclude that traditional clipped-artery harvesting of the LIMA disrupts the integrity of the luminal endothelium and hence decreases NO production. We believe that the modified nonclipped-artery harvesting technique better preserves endothelial integrity and function. However, the long-term effects of both techniques on survival and patency rates remain to be determined.

Footnotes

Address for reprints: Mustafa Buyukates, MD, Department of Cardiovascular Surgery, School of Medicine, Zonguldak Karaelmas University, Kozlu–Zonguldak 67600, Turkey. E-mail: moc.oohay@setakuyubafatsum

References

1. Loop FD, Lytle BW, Cosgrove DM, Stewart RW, Goormastic M, Williams GW, et al. Influence of the internal-mammary-artery graft on 10-year survival and other cardiac events. N Engl J Med 1986;314:1–6. [PubMed]
2. Lytle BW, Loop FD, Cosgrove DM, Taylor PC, Goormastic M, Peper W, et al. Fifteen hundred coronary reoperations. Results and determinants of early and late survival. J Thorac Cardiovasc Surg 1987;93:847–59. [PubMed]
3. Grondin CM, Campeau L, Lesperance J, Enjalbert M, Bourassa MG. Comparison of late changes in internal mammary artery and saphenous vein grafts in two consecutive series of patients 10 years after operation. Circulation 1984;70(3 Pt 2):I208–12. [PubMed]
4. Fitzgibbon GM, Kafka HP, Leach AJ, Keon WJ, Hooper GD, Burton JR. Coronary bypass graft fate and patient outcome: angiographic follow-up of 5,065 grafts related to survival and reoperation in 1,388 patients during 25 years. J Am Coll Cardiol 1996;28:616–26. [PubMed]
5. Grondin CM, Campeau L, Thornton JC, Engle JC, Cross FS, Schreiber H. Coronary artery bypass grafting with saphenous vein. Circulation 1989;79(6 Pt 2):I24–9. [PubMed]
6. Cameron A, Davis KB, Green G, Schaff HV. Coronary bypass surgery with internal-thoracic-artery grafts–effects on survival over a 15-year period. N Engl J Med 1996;334:216–9. [PubMed]
7. Berkowitz HD, Fox AD, Deaton DH. Reversed vein graft stenosis: early diagnosis and management. J Vasc Surg 1992; 15:130–42. [PubMed]
8. Kaufer E, Factor SM, Frame R, Brodman RF. Pathology of the radial and internal thoracic arteries used as coronary artery bypass grafts. Ann Thorac Surg 1997;63:1118–22. [PubMed]
9. Ruengsakulrach P, Sinclair R, Komeda M, Raman J, Gordon I, Buxton B. Comparative histopathology of radial artery versus internal thoracic artery and risk factors for development of intimal hyperplasia and atherosclerosis. Circulation 1999;100(19 Suppl):II139–44. [PubMed]
10. Garg UC, Hassid A. Nitric oxide-generating vasodilators and 8-bromo-cyclic guanosine monophosphate inhibit mitogenesis and proliferation of cultured rat vascular smooth muscle cells. J Clin Invest 1989;83:1774–7. [PMC free article] [PubMed]
11. Mayer B, Hemmens B. Biosynthesis and action of nitric oxide in mammalian cells [published erratum appears in Trends Bio-chem Sci 1998;23:87]. Trends Biochem Sci 1997;22: 477–81. [PubMed]
12. Gaudino M, Toesca A, Maggiano N, Pragliola C, Possati G. Localization of nitric oxide synthase type III in the internal thoracic and radial arteries and the great saphenous vein: a com-parative immunohistochemical study. J Thorac Cardiovasc Surg 2003;125:1510–5. [PubMed]
13. Grapow MT, Konerding MA, Muller-Schweinitzer E, Bernet F, Matt P, Reineke DC, Zerkowski HR. Protecting the endothelial integrity of internal thoracic arteries. Thorac Cardiovasc Surg 2005;53:352–7. [PubMed]
14. Grapow MT, Kern T, Reineke DC, Brett W, Bernet F, Rue-ter F, et al. Improved endothelial function after a modified harvesting technique of the internal thoracic artery. Eur J Car-diothorac Surg 2003;23:956–61.
15. Saxena P, Mejia R, Tam R. Hydrodissection technique of harvesting left internal thoracic artery. Ann Thorac Surg 2005; 80:355–6. [PubMed]
16. Loop FD. Internal-thoracic-artery grafts. Biologically better coronary arteries. N Engl J Med 1996;334:263–5. [PubMed]
17. Marx R, Jax TW, Plehn G, Schannwell CM, Horlitz M, Klein RM, et al. Morphological differences of the internal thoracic artery in patients with and without coronary artery disease–evaluation by duplex-scanning. Eur J Cardiothorac Surg 2001;20:755–9. [PubMed]
18. Groves JT, Wang CC. Nitric oxide synthase: models and mechanisms. Curr Opin Chem Biol 2000;4:687–95. [PubMed]
19. Tsui JC, Souza DS, Filbey D, Karlsson MG, Dashwood MR. Localization of nitric oxide synthase in saphenous vein grafts harvested with a novel “no-touch” technique: potential role of nitric oxide contribution to improved early graft patency rates. J Vasc Surg 2002;35:356–62. [PubMed]
20. Buttery LD, Chester AH, Springall DR, Borland JA, Michel T, Yacoub MH, Polak JM. Explanted vein grafts with an intact endothelium demonstrate reduced focal expression of endothelial nitric oxide synthase specific to atherosclerotic sites. J Pathol 1996;179:197–203. [PubMed]
21. Ku DD, Zaleski JK, Liu S, Brock TA. Vascular endothelial growth factor induces EDRF-dependent relaxation in coronary arteries. Am J Physiol 1993;265(2 Pt 2):H586–92. [PubMed]
22. Broeders MA, Doevendans PA, Maessen JG, van Gorsel E, Egbrink MG, Daemen MJ, et al. The human internal thoracic artery releases more nitric oxide in response to vascular endothelial growth factor than the human saphenous vein. J Thorac Cardiovasc Surg 2001;122:305–9. [PubMed]
23. Liu ZG, Ge ZD, He GW. Difference in endothelium-derived hyperpolarizing factor-mediated hyperpolarization and nitric oxide release between human internal mammary artery and saphenous vein. Circulation 2000;102(19 Suppl 3):III296–301. [PubMed]
24. He GW, Liu ZG. Comparison of nitric oxide release and endothelium-derived hyperpolarizing factor-mediated hyperpolarization between human radial and internal mammary arteries. Circulation 2001;104(12 Suppl 1):I344–9. [PubMed]
25. Pollock JS, Nakane M, Buttery LD, Martinez A, Springall D, Polak JM, et al. Characterization and localization of endothelial nitric oxide synthase using specific monoclonal antibodies. Am J Physiol 1993;265(5 Pt 1):C1379–87. [PubMed]
26. Dikranian K, Trosheva M, Nikolov S, Bodin P. Nitric oxide synthase (NOS) in the human umbilical cord vessels. An immunohistochemical study. Acta Histochem 1994;96:145–53. [PubMed]
27. Gaudino M, Toesca A, Nori SL, Glieca F, Possati G. Effect of skeletonization of the internal thoracic artery on vessel wall integrity. Ann Thorac Surg 1999;68:1623–7. [PubMed]
28. Ueda T, Taniguchi S, Kawata T, Mizuguchi K, Nakajima M, Yo-shioka A. Does skeletonization compromise the integrity of internal thoracic artery grafts? Ann Thorac Surg 2003;75: 1429–33. [PubMed]
29. Yoshikai M, Ito T, Kamohara K, Yunoki J. Endothelial integrity of ultrasonically skeletonized internal thoracic artery: mor-phological analysis with scanning electron microscopy. Eur J Cardiothorac Surg 2004;25:208–11. [PubMed]
30. Deja MA, Wos S, Golba KS, Zurek P, Domaradzki W, Bachowski R, Spyt TJ. Intraoperative and laboratory evaluation of skeletonized versus pedicled internal thoracic artery. Ann Thorac Surg 1999;68:2164–8. [PubMed]
31. Goff SG, Wu HD, Sauvage LR, Usui Y, Wechezak AR, Coan DE, et al. Differences in reendothelialization after balloon catheter removal of endothelial cells, superficial endarterectomy, and deep endarterectomy. J Vasc Surg 1988;7:119–29. [PubMed]
32. Sasajima T, Wu MH, Shi Q, Hayashida N, Sauvage LR. Effect of skeletonizing dissection on the internal thoracic artery. Ann Thorac Surg 1998;65:1009–13. [PubMed]

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