J Thorac Cardiovasc Surg 1995;110:81-88
© 1995 Mosby, Inc.
Received for publication Feb. 24, 1994. Accepted for publication Nov. 7, 1994. Address for reprints: Pekka Rainio, MD, PhD, Department of Surgery, Oulu University Central Hospital, Kajaanintie 50, SF-90220 Oulu, Finland.
Ultrastructural changes in myocardial tissue were studied in 21 patients undergoing elective aorta-coronary bypass operation. The patients were randomized into two groups, with 10 of them receiving continuous retrograde warm and 11 continuous retrograde mild hypothermic blood cardioplegia. Biopsy specimens for electron microscopy were taken from the apical part of the left ventricle before and at the end of the aortic crossclamp period and after reperfusion of the myocardium. The ultrastructural changes were analyzed with use of a semiquantitative scoring system and classified as mild, moderate, or severe. Slight ultrastructural changes were found in both groups even before the aortic crossclamp period. At the end of the aortic crossclamp period the most prominent ultrastructural changes were mitochondrial swelling, damage of capillary endothelium, and clearing of the nucleoplasm or margination of chromatin, but some enlargement in intercalated discs was also discernible. Reperfusion of the myocardium for 15 minutes somewhat further increased the overall score of the ultrastructural changes. Two patients in the warm cardioplegia group had a perioperative myocardial infarction, and this may be one reason for the higher postoperative creatine kinase MB efflux in this patient group. Despite this finding, no major differences in the ultrastructural changes between the two cardioplegia groups could be observed. We conclude that only mild to moderate and principally reversible ultrastructural changes occur in myocardium during continuous retrograde warm and mild hypothermic blood cardioplegia for coronary bypass operation. (J THORACCARDIOVASCSURG1995;110:81-8)
Ultrastructural changes in the myocardium during heart operations have been detected in experimental studies, 1, 2 as well as in human populations. 3 After ischemia of only 10 to 15 minutes, swelling of mitochondria and margination of nuclear chromatin may be observed. 1 Mitochondria are the first cellular components to show the effect and degree of ischemia. Endothelial cells in the microvasculature of the myocardium are also highly vulnerable to ischemic injury. 4 Loss of pinocytotic vesicles and thinning of the endothelium are the first signs of endothelial injury. The "no-reflow" phenomenon, which involves cellular swelling within and around the capillaries, causing occlusion of the lumen, worsening of perfusion, and, finally, ischemic cell damage, was first ascertained in brain capillaries. 5 The same phenomenon has also been observed in the heart. 4
Adequate protection of the myocardium during heart operations is the major prerequisite for a good outcome. The protective effects of cardioplegic solutions differ, and even very severe cell damage has been described. 2 Myocardial areas distal to complete coronary artery obstructions are poorly protected by antegrade cardioplegia, and retrograde administration of cardioplegic solution through the coronary sinus has emerged as an attractive alternative for these patients. The purpose of this study was to investigate the ultrastructural changes that take place during continuous retrograde warm or mild hypothermic blood cardioplegia during coronary artery bypass operations.
All patients had stable angina pectoris (New York Heart Association [NYHA] class III), three-vessel disease shown by coronary angiography, and normal left ventricular (LV) function as assessed by left ventriculography (Table I). Before operation, the patients signed a consent form approved by the Ethical Committee at the Oulu University Hospital.
The blood cardioplegia was diluted in a 7:1 (Dideco D 720 blood cardioplegia delivery set, Dideco S.P.A., Mirandola, Italy) ratio with a solution containing aspartate monohydrate and glutamate monohydrate in 300 ml of water (13 mmol/L each), 250 ml of 5% glucose, 150 ml of tribonate (TRIS bicarbonate; Tribo rat, Kabi, Sweden), 125 ml of citrate-phosphate-dextrose, and 40 ml of KCl (2 mol/L).
After aortic crossclamping warm high-potassium cardioplegic solution was administered into the aortic root for 2 minutes at a flow rate of 200 ml/min, and as soon as the heart was arrested the delivery of cardioplegia was switched to a retrograde method and continued with low-potassium cardioplegia in a randomized manner (37º C or 28º C) via a self-inflating coronary sinus cannula (catalog number Rc-014T, Research Medical, Inc, Midvale, Utah). The cannula was inserted blindly into the coronary sinus and care was taken to avoid mechanical blockade of the distal branches of sinus and to maximize right ventricular preservation. The coronary sinus pressure was continuously monitored and kept less than 40 mm Hg, and the flow rate was adjusted to approximately 125 ml/min. The retrograde cardioplegia was continued throughout the operation with the exception of occasional short interruptions during the suturing of the distal anastomoses. The ischemic times were comparable in the two study groups (Table I).
The distal anastomoses were completed in the order of importance according to surgeon's preference, except that the left internal mammary artery (used in all patients) was anastomosed last. The proximal vein anastomoses were finished before aortic declamping.
Reduced doses of aprotinin were given to six patients (all together 3 million units), three patients in both groups. These patients had used aspirin until the operation.
Blood samples for creatine kinase (CK) measurements were collected at 4-hour intervals for 48 hours after operation. CK levels were analyzed by a technique standardized according to Scandinavian requirements. CK-MB levels were determined by electrophoresis. Hemodynamic parameters were determined by arterial blood pressure monitoring and catheterization with a Swan-Ganz catheter (Baxter Healthcare Corp., Edwards Division, Santa Ana, Calif.).
The first transmural needle biopsy (Tru-Cut needle, William Schmidt, Inc., Valencia, Calif.) was done before induction of cardiac arrest and aortic crossclamping. The second biopsy was done before aortic declamping and the third after 15 minutes of reperfusion during cardiopulmonary bypass. All the biopsy samples were taken from the apical parts of the left ventricular wall, which was macroscopically normal. For technical reasons, the first biopsy sample was not available for analysis in two patients in the mild hypothermic group, the second biopsy sample from two patients in the warm cardioplegic group, and the third biopsy sample from one patient in both groups. The tissue plug specimens were immediately fixed with 2.5% glutaraldehyde in 0.1 mol/L phosphate buffer, pH 7.4, for 1 hour and then postfixed in 1% OsO4 in 0.1 mol/L phosphate buffer, pH 7.4, for 1 hour, dehydrated in acetone, and embedded in Epon LX 112 fixative. Semithin sections stained with toluidine blue were prepared from all tissue samples. The sections were cut with a Reichert Ultracut E-ultramicrotome (Leica, Inc., Buffalo, N.Y.) and examined under a Philips 410 LS transmission electron microscope (Philips Electronic Instruments, Inc., Mahwah, N.J.), with use of an acceleration voltage of 60 kV.
The intercellular junctions (intercalated discs) intracellular and extracellular edema, mitochondria, capillaries, nuclei and myofibrils were analyzed separately in each biopsy specimen by a semiquantitative method with scoring from 0 (unchanged) to 3 (severe alterations). The electron microscopist was blinded as to the sequence of the specimens and the group to which the patients belonged. A total score of all ultrastructural changes less than 5 was defined as mild damage, scores ranging from 5 to 10 as moderate, and scores exceeding 10 as severe ultrastructural damage.
Statistical analysis was done with use of the SPSS statistical package program (SPSS, Inc., Chicago, Ill.). Unpaired Student's test and Fisher's exact test were used to compare the clinical characteristics and the patient groups. Analysis of variance for repeated measurements was used to test the time-dependent ultrastructural changes in both cardioplegia groups.
Clinical data (number of patients, age, NYHA classification, ejection fraction) and operative data (crossclamp time, cardiopulmonary bypass time, ischemia time, number of distal anastomoses) were comparable between the two patient groups (Table I). Coronary stenoses were analyzed separately, and in this respect the groups were also comparable (Table II). There were no significant differences in the preoperative and postoperative values of the cardiac index or the calculated left and right stroke work indexes (not shown). The postoperative CK-MB efflux in both groups is shown in Fig. 1 and this was significantly (p = 0.005) higher in the warm cardioplegia group. Two patients in the warm cardioplegia group had CK-MB levels greater than 60 IU/L, but no new Q waves were observed.
The mean ischemic times were 5.2 ± 2.8 (5.3%) minutes and 8.4 ± 8.3 (8.3%) minutes in the warm and mild hypothermic groups, respectively (difference not significant). The ischemic times did not correlate with the score values of ultrastructural changes in either of the study groups.
The ultrastructural changes in the third biopsy samples taken after release of the crossclamp were somewhat more prominent than those in the second biopsy samples taken at the end of aortic crossclamping.
The score values in patients who received aprotinin during the operation did not differ from the values in patients who did not receive aprotinin.
In this study 21 patients with coronary artery disease undergoing elective coronary artery bypass grafting were randomized into two groups on the basis of the temperature of the retrograde blood cardioplegia solution. The ultrastructural changes that occurred in the myocardium during the operations were subsequently analyzed. The cell damage during aortic crossclamping (short-time ischemia) and reperfusion (15 minutes after aortic declamping) was compared to that observed in biopsy samples taken before aortic crossclamping. All the biopsy samples were taken from macroscopically normal left anterior myocardium.
It has been shown earlier that the results of semiquantitative analysis correlate well with those of morphometric analysis, 6 making it a useful nonparametric method for studying ischemic cell damage. Mitochondria have been suggested to be better preserved during warm retrograde than cold antegrade cardioplegia, 7 but no differences have been found between blood antegrade or intermittent cardioplegia. 8 We found the most distinct ultrastructural changes in the mitochondria of myocytes and the endothelium. In some cases mitochondria showed a honeycomb appearance and zigzag profiles of cristae, but they were not totally destroyed. One patient in the mild hypothermic group showed the development of intramitochondrial Jennings bodies, which are granular densities of calcium phosphate caused by permanent ischemia. 1 Crystalline inclusions and rodlike bodies were also found in one patient, but they can be regarded as a nonspecific feature of cell damage. 9, 10
It has been suggested that low protein-denaturation embedding techniques do not lead to extraction of mitochondrial proteins and therefore the crista membranes are preserved better than after osmium tetroxide fixation and embedding. 11 According to our experience, however, biopsy specimens postfixed in 1% osmium tetroxide, dehydrated in acetone, and embedded in Epon fixative maintain their subcellular units relatively well and therefore can be used for semiquantitative analysis. In support for this, we found only minimal ultrastructural changes in the myocardium of two patients without coronary artery disease undergoing cardiac operation because of secundum-type atrial septal defect (results not shown).
In the second biopsy specimens, we were able to find well-preserved foci and also poorly preserved foci with nearly total endothelial damage and intraluminal myelin figures and myofibrillary lysis suggesting a certain degree of heterogeneity in the protection of the myocardial ultrastructure (e.g., subendocardium versus subepicardium), at least under the conditions of the present study.
All the patients had three-vessel coronary artery disease and were functionally classified to have NYHA class III symptoms. Consequently these patients had had repeated ischemia/reperfusion sequences before operation as a result of repeated anginal attacks. Thus it is not surprising that slight endothelial damage and increasing intramitochondrial swelling (Figs. 4 and 5) were noticed even in the first biopsy specimens taken before aortic crossclamping. The no-reflow phenomenon is associated with reperfusion injury in open heart operations. 4 On the other hand, it may well be that some of the changes, such as cellular edema, increased collagen, and lipofuscin accumulation might have been a result of chronic "hibernation" of the myocardium, which is known to be associated with certain ultrastructural changes. 12-14
Aprotinin is a protease inhibitor that might protect the myocardium from the adverse effects of ischemia by suppressing the release of lysosomal enzymes. The mitochondria and myofibrils of isolated canine hearts given aprotinin are better preserved than those without aprotinin but, on the other hand, aprotinin may impair the functional recovery of the left ventricle. 15 Both of our cardioplegia groups included three patients who received aprotinin during the operation, but no differences in their postoperative recoveries could be detected, nor were their mitochondrial changes different from those of the patients not given aprotinin.
Significant differences of CK effluxes after the operations were noticed in favor of the mild hypothermic group. Two patients from the normothermic cardioplegia group had a perioperative myocardial infarct, which increased the CK-MB value, and this may be one reason for the observed difference. There was no perioperative or 30-day mortality in this series. It was interesting to note that the two patients with perioperative myocardial infarctions also showed the most severe myofibrillar changes in the biopsy specimens.
We conclude that continuous retrograde blood cardioplegia protects myocardium sufficiently to prevent extensive cellular damage, inasmuch as only slight to moderate ultrastructural changes were observed during coronary bypass operations of considerable length. No significant differences in the myocardial ultrastructure could be detected in the normothermic or mild hypothermic cardioplegia groups.
From the Departments of Surgery,a Anesthesiology,c Internal Medicine,d and Pathology,b Oulu University Hospital, Oulu, Finland.
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