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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.
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.
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.
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 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.
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.
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).
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).
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.
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