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Ann Thorac Surg 2004;78:1319-1325
© 2004 The Society of Thoracic Surgeons
a Clinic of Cardiovascular Surgery, Türkiye Yuksek Ihtisas Hospital, Ankara, Turkey
b Clinic of Cardiothoracic Surgery, Ankara Numune Hospital, Ankara, Turkey
c Department of Anatomy, Hacettepe University Faculty of Medicine, Ankara, Turkey
d Department of Biochemistry, Hacettepe University Faculty of Medicine, Ankara, Turkey
Accepted for publication March 15, 2004.
* Address reprint requests to Dr Ozisik, 23 Cad. Kirkkonaklar Mah. Simkent Sitesi, 2 Blok, No 6, 13, 06610 Ankara, Turkey.
sozisik2002{at}yahoo.com
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Abstract |
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METHODS: Radial artery samples from 4 groups of patients (10 samples from each group) who underwent coronary artery surgery were evaluated. In groups I and II, radial arteries were stored in a solution containing 100 mL patients' own heparinized oxygenated blood and 5 mg nitroglycerine. The grafts were distended in group II. In groups III and IV, side branches of the grafts were occluded and left in situ until the coronary anastomosis. In group III, the distal end of the graft was also occluded, while in group IV, both ends were open.
RESULTS: The least degree of endothelial damage and disruption of arterial wall integrity were in group IV according to electron microscopic evaluation. The grafts in group III were relatively in good condition compared to groups I and II. Tissue lipid peroxidation was also lowest in group IV. Tissue lipid peroxide levels correlated with ischemic preservation period of the radial artery (r = 0.426). Total semiquantitative transmission electron microscopic scores of the radial artery samples correlated with the lipid peroxide levels (r = 0.581) and ischemia times (r = 0.734).
CONCLUSIONS: All arterial grafts, including the radial artery that will be used for coronary artery surgery should not be left ischemic during harvesting to prevent endothelial damage. Ischemia of the conduits for coronary artery grafting can be prevented by leaving them in their anatomic position until the distal coronary artery anastomosis.
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Introduction |
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TopAbstractIntroductionMaterial and MethodsResultsCommentReferences |
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Arterial grafts have been used increasingly in coronary artery bypass surgery in the last three decades [2]. Arterial grafts, especially the internal mammary artery (IMA), have been proven to have superior patency rates over vein grafts [3]. The RA grafts were introduced as coronary bypass conduits in 1973 by Carpentier and colleagues [4]. Unfortunately, high occlusion rates were reported and the utilization of RA was almost completely abandoned [5, 6]. After control angiograms of patients who were operated more than 10 years ago with patent RA grafts had been obtained, Acar and colleagues reintroduced the use of RA for coronary bypass in 1992 [7]. In our clinic, RA is a preferred graft, when complete arterial revascularization is intended, especially for patients younger than 60 years old.
Early graft failures were attributed to graft vasospasm and premature occlusions to intimal hyperplasia. In the second era of RA utilization, to prevent graft vasospasm and achieve better long-term patency, harvesting techniques were clearly refined. The storage media was scrutinized, and the systemic and topical vasodilator drugs were introduced to routine practice [8–11]. Disruption and disintegration of endothelium of this type III muscular artery during harvesting could prevent endothelium dependent relaxation and probably cause early graft failure. Trauma during harvesting is the major cause of endothelial damage. Storing the graft in a sanguineous media, with or without vasodilators, may result in ischemic damage of the endothelium [10]. It was reported that storage in heparinized and oxygenated blood caused increased vessel reactivity to vasoconstrictors in RA due to the inhibition of nitric oxide released by oxyhemoglobin [12].
To evaluate the effects of storage media and harvesting techniques, a double-blind, randomized, and prospective study was conducted. The purpose of our study was to determine the least damaging RA harvesting method. We believe that a harvesting technique with less damage would eventually improve early and late outcomes of patients. Specimens of radial arteries from four groups of patients were examined with transmission electron microscopy (TEM) to document the endothelial changes with a semiquantitative scoring method. The grafts were prepared with four different techniques. The results were compared in terms of the level of damage to endothelium. The lipid peroxidation levels of tissue samples were also evaluated as a secondary indicator of tissue ischemia, and a correlation was sought with the electron microscopic morphologic changes.
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Material and Methods |
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Patients
The patients who were enrolled in the study were randomly assigned to the groups and RA was harvested according to the predefined technique for each group. Four study groups were formed, according to the harvesting technique and the storage media of the RA graft. There were 10 patients in each group. The mean age of the patients was 48.1 ± 6.6 (range, 37 to 59). Only 2 patients were female (5%). Eight patients were diabetic (20%), 34 patients were smokers or ex-smokers (85%). Hypertension was present in 12 (30%) patients. Family history of atherosclerotic cardiac diseases was elicited in 20 (50%) patients. Seventy-five percent (n = 30) of the patients who were included in the study were hyperlipidemic, and five patients (12.5%) were morbid obese.
Complete arterial revascularization was performed in 12 of the patients. Greater saphenous vein was also prepared instead of a second IMA when the patient was diabetic or more than three grafts were needed. Right IMA was used in 6 patients. The RA graft was utilized in a sequential fashion to more than one target vessel in three patients. Greater saphenous vein and at least one mammary artery were used in 28 patients in addition to RA. None of the RA grafts was wasted. Two of the left internal mammary arteries were discarded due to insufficient flow.
Radial artery grafts with accompanying veins were harvested. Side branches were occluded with small hemostatic clips. Electrocautery was not used. In groups I and II, RAs were prepared carefully and stored in a solution containing 100 mL of patients' own heparinized oxygenated (arterial) blood and 5 mg of nitroglycerine. The grafts were not distended with pressure in group I, but the grafts in group II were distended with the same solution just before the performance of coronary artery anastomosis. Papaverin was not used in these two groups. In groups III and IV radial arteries were prepared but left in situ until the time of coronary anastomosis. The distal end was occluded with hemostatic clips in group III, but the proximal end was not occluded or dissected. In group IV, proximal and distal ends were neither occluded nor dissected. The grafts were left in their anatomic places until the performance of coronary artery anastomoses. During this period, the grafts were also covered with gauze soaked with isotonic saline and papaverin solution. A solution containing 8 mL of isotonic saline and 50 mg of papaverin was also sprayed on the grafts before being covered with gauze. No extra distention was applied.
Data Collection
In addition to the age, sex, and coronary artery disease, preoperative history of smoking, atherosclerotic trait in family, history of hypertension, presence of diabetes mellitus, and hyperlipidemia were recorded. Preoperative levels of blood urea, total cholesterol, high density lipoprotein (HDL) cholesterol, and low density lipoprotein (LDL) cholesterol were obtained. As operative data, total cardiopulmonary bypass time, cross-clamp time, number of bypassed coronary arteries, grafts, and degree of operative hypothermia during cardiopulmonary bypass were documented. Tissue samples of RAs were collected for TEM evaluation and measurement of tissue lipid peroxide level, which is a strong marker of tissue ischemia [13], just before the proximal anastomosis of the conduit to the ascending aorta. The proximal end of the RA was always used for the anatomosis to the aorta. Tissue samples were always taken from the most proximal end of the graft from every patient enrolled in the study. Ischemia time was defined as the period starting with the dissection of the proximal end from the brachial artery after preparation, until its proximal anastomosis to the aorta. In groups III and IV, the proximal end of the RA was dissected just before the coronary artery anastomosis.
Transmission Electron Microscopic Evaluation
The grafts were measured; an extra length of 0.5 cm of RA graft from the proximal end was divided just before the proximal anastomosis on the aorta. These arterial samples were immediately immersed into 2.5% of glutaraldehyde solutions. The specimens were fixed in glutaraldehyde for 24 hours, washed in phosphate buffer (pH: 7.4), postfixed in 1% of osmium tetroxide in phosphate buffer (pH: 7.4), and dehydrated in increasing concentrations of alcohol. The tissues were then washed with propylene oxide and embedded in epoxy-resin embedding media. Semithin sections about 2 µm in thickness and ultrathin sections about 60 nm in thickness were cut with a glass knife on an LKB-Nova (Nova, Bromma, Sweden) ultramicrotome. Semithin sections were stained with methylene blue and examined by a Nikon Optiphot (Nikon, Tokyo, Japan) light microscope. Ultrathin sections were collected on copper grids, stained with uranyl acetate and lead citrate and examined with a Joel JEM 1200 EX (Joel, Tokyo, Japan) TEM. During the electron microscopic evaluations, the histopathologist (MFS) was blinded. In addition to a general description of morphologic changes of tissue samples, a semiquantitative method of scoring was also applied for every artery. This scoring method is original and developed by the histopathologist himself (Appendix). In brief, the method consisted of an evaluation of every slide for 3 individual morphologic changes: (a) structure of endothelial cells; (b) degree of tissue edema; and (c) morphologic changes of mitochondria in endothelial cells. For all criteria, "0" score meant normal and "3" defined the most degenerated. Ten different areas on slides for every artery were evaluated and scored. Four average scores, which were endothelial score, edema score, mitochondrial score, and the sum of these three scores, were achieved for every sample. Mean scores were calculated for each group.
Lipid Peroxidation Assay
The tissue samples of the RAs were thoroughly cleansed of blood and were immediately frozen and stored in a –20°C freezer for assays of malondialdehyde. The level of lipid peroxidation in the RA was determined using the method of Mihara and Uchiyama [14]. Tissues were homogenized in 10 volumes (w/v) of cold phosphate buffer (pH: 7.4). A half milliliter of homogenate was mixed with 3 mL of 1 percentage H3PO4. After the addition of 1 mL of 0.67% thiobarbituric acid, the mixture was heated in boiling water for 45 minutes. The color was extracted into n-butanol, and the absorption at 532 nm was measured. Using tetramethoxypropane as the standard, tissue lipid peroxidation levels were calculated as nanomole per gram of wet tissue. During these assays, the biochemist (KK) was also blinded to the sample and the patient.
Statistical Analysis
Data were analyzed using commercially available statistical software packages. The nominal data were expressed as percentages and compared with the 
2 test. All continuous variables were given as mean ± standard deviation and compared with analysis of variance (ANOVA). The scores achieved from electron microscopic evaluation were accepted as continuous data and included in univariate and multivariate analyses. Tukey's posthoc tests were used. All statistics were selectively made for diabetic patients before any further tests being performed to overcome any bias due to the patient characteristics. The presence of diabetes did not have any effect on any factor evaluated. Correlation was sought among the operative data, semiquantitative electron microscopic scores, and tissue lipid peroxide levels using the Pearson method of analysis. Linear regression analysis for the total electron microscopic score was also performed with using the "enter" method. The p values less than 0.05 were considered as significant for all results.
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Results |
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TopAbstractIntroductionMaterial and MethodsResultsCommentReferences |
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The differences between measured tissue lipid peroxide (TLPO) levels were statistically significant in both univariate and multivariate analysis. The TLPO was the lowest in group IV, and statistically different from the other groups. The TLPO measurements were not different among groups I, II, and III.
All operative data were assessed to find any existing correlations between the variables. The most striking correlation was found between the TLPO levels and RA ischemia duration (r = 0.426, p = 0.006). (Fig 1). There were correlations which could be expected beforehand. These kinds of correlations were found between cardiopulmonary bypass and cross-clamp durations. The RA ischemia times and cardiopulmonary bypass (r = 0.702, p < 0.001) and cross-clamp duration (r = 0.696, p < 0.001).
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Transmission Electron Microscopic Findings (Semiquantitative Scoring)
Results of semiquantitative electron microscopic evaluation are given in Table 2. The highest total score was achieved in group II. The scores of group II were significantly higher than others (p < 0.001). Posthoc tests for ANOVA also revealed the same result. Group I also had higher scores when compared with groups III and IV. The only difference observed between TEM evaluation scores of groups III and IV was in the scores of the mitochondrial changes. The scores given for the mitochondrial changes in endothelial cells of group III were significantly higher than the scores of group IV (p = 0.012). There were correlations with the mean total scores of the groups and the TLPO levels (r = 0.581, p < 0.001), as well as the ischemia time (r = 0.734, p < 0.001) (Figs 2 and 3). Linear regression analysis revealed that ischemia time (p < 0.001) and TLPO levels (p = 0.009) were the significant predictors of total electron microscopic scores.
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Group III
The edema of the endothelial and subendothelial layer were significantly less in the arteries of this group, when it was compared to those in groups I and II. No pathology was observed in the continuity of the endothelial layer. There was subendothelial edema in scattered areas but it was not prominent. Lesser degree of cytoplasmic mitochondrial swelling was noted. Edema present in tunica media and adventitia of the artery was found less significant when it was compared to those in groups I and II (Figs 4E and 4F).
Group IV
The degree of edema of the endothelial and subendothelial layer was negligible, and the endothelial layer was intact in this group. Subintimal edema was not observed. Mitochondrial swelling was rarely encountered, and was less prominent when it was compared to other groups. Edema of the tunica media and adventitia was also negligible (Figs 4G and 4H).
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Comment |
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TopAbstractIntroductionMaterial and MethodsResultsCommentReferences |
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-adrenergic receptors, predominantly. The combined thickness of media and intima of the RA is the greatest among the arterial grafts [1]. These properties of the RA are probably the reason for its major disadvantage, which is the propensity to vasospasm. Enhanced reactivity of the RA has been well documented both clinically and in vitro studies. Despite this major drawback, the RA is still an attractive arterial conduit for coronary artery bypass surgery for several reasons: (1) it is easy to harvest in parallel with the IMA; (2) it is long enough to be used as a graft for any coronary artery; (3) it is large enough in caliber to match the most of the coronary arteries; and (4) it is easy to handle due to its thick muscular wall [8]. Systemic and topical vasodilator drugs are used in routine practice to prevent early graft spasm [8, 9]. Skeletinization of the artery with an ultrasonic scalpel is offered for better early and late term patency [11, 15]. We believe that if endothelial integrity of the radial artery can be preserved along with the application of systemic and topical drugs, it will provide better early and late patency rates of the radial artery grafts. Endothelium is described as an organ that controls the vasoreactivity of vessels, with many hormonal and secretory properties. In the present study, we have compared the integrity of RA ultrastructure with TEM in four different harvesting techniques. The disruption of the integrity of intima and media, which was shown in our study, seems to be related to ischemia during coronary artery grafting which may in turn affect the long-term outcomes of patients. Furthermore, the levels of TLPO were found to be correlated with the ischemia time, which is consistent with a previous study [13] that reported the levels of TLPO could be a good indicator of ischemic tissue damage (Fig 1). The correlations found in our study between TLPO levels and ischemia time and electron microsopic changes indicate that leaving the radial artery graft in situ until the performance of coronary artery anastomosis preserves the integrity of its endothelium.
The work of Cable and colleagues [16] showed that baseline release of vasoactive substances of the IMA and RA was similar, but the release of the RA was reduced in stimulated conditions, compared to the IMA. Cyclic guanosine monophosphate (cGMP) levels were similar in both arteries in baseline conditions, but with stimulation, the IMA produces significantly higher levels of cGMP. Moreover, vasodilation observed with acetylcholine was much more pronounced in the IMA. The difference between the vasodilatatory response to nitric oxide donor drugs of the IMA and RA was not evident [16]. Cable and colleagues [16] stated that reduced production of endothelium derived relaxing factors suggested diminished endothelial regulation of vascular smooth muscle in the RA compared to the IMA.
Attenuation of vasoreactivity of the RA may outweigh early risks caused by vasospasm of the RA and hypoperfusion of the myocardial areas. Intactness of the endothelial layer may probably preserve vasoreactivity of the arterial conduit. It may be discussed whether this is a desired option for early postoperative period. We believe that an intact endothelial layer reacts with all its capacity (by means of endothelium-derived nitric oxide, prostaglandin I2, and endothelium-derived hyperpolarizing factor) to supply blood to ischemic myocardium, which reveals the need for secretion of certain metabolic and locally acting substances. Therefore, the integrity of the endothelial layer is important for the clinical outcome of the patient.
In conclusion, we believe that the RA should not be left ischemic during harvesting to prevent endothelial damage, which applies to all arterial grafts, including the IMA. Ischemia of the conduits that are planned to be used for coronary artery grafting can be prevented by leaving them in their anatomic position with both ends open, until the distal coronary artery anastomosis.
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Appendix |
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(A) Structure of endothelial cells: "0", ultrastructurally in normal thickness; "1", endothelial cells which have a decrease in their thickness; "2", presence of discontinuities in the endothelial wall; "3", presence of completely damaged endothelial layer.
(B) Degree of tissue edema: 0, no edema in the wall of the arteries; 1, edema in the endothelial layer; 2, edema in the endothelium and subendothelium; 3, edema in the endothelium, subendotheliun, tunica media, and tunica adventitia.
(C) Morphologic changes of mitochondria in endothelial cells: 0, normal; 1, mild degree of swelling (mitochondrion with prominent cristae); 2, swollen mitochondrion (cloudy swelling); 3, amorphous material deposition in the mitochondrion.
Ten different areas on slides for every artery were evaluated and scored. Four average scores were achieved for every sample: endothelial score, edema score, mitochondrial score, and the sum of the three scores. Mean scores were calculated for every group.
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References |
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TopAbstractIntroductionMaterial and MethodsResultsCommentReferences |
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