Leukocyte Depletion Attenuates Reperfusion Injury in Patients With Left Ventricular Hypertrophy
Background Reperfusion injury can occur after a long period of aortic cross-clamping in patients with left ventricular hypertrophy during open-heart surgery, even with the most up-to-date techniques of myocardial protection. In the present study, we examined whether leukocyte depletion as an adjunct to terminal blood cardioplegia (LDTC) attenuates reperfusion injury in patients with left ventricular hypertrophy (LV mass, >300 g; left ventricular end-systolic volume index, >100 mL/m2) in a group of 30 patients undergoing aortic valve replacement.
Methods and Results We used basic cold potassium crystalloid cardioplegic solution. Terminal blood cardioplegic solution (TC) or LDTC was accomplished by mixing a cold potassium crystalloid cardioplegic solution with warm arterial blood obtained through cardiopulmonary bypass and administered to the aortic root for the first 10 minutes of reperfusion. During delivery of LDTC, warm arterial blood was passed through a leukocyte-removal filter. Patients were randomized into one of three groups for reperfusion: whole blood (WB) (n=10), TC (n=10), and LDTC (n=10). Left ventricular biopsies were obtained before ischemia, at the end of ischemia, and 15 minutes after reperfusion. Semiquantitative scoring for ultrastructural alterations indicated that the LDTC group achieved significantly better recoveries of both scores at reperfusion for myocyte damage and for endothelial cell damage of capillaries than did the WB and TC groups. The LDTC group had significantly fewer neutrophils adhering to endothelial cells at reperfusion and a lower level of malondialdehyde derived from myocardium than did the WB and TC groups. Regarding the clinical data, the LDTC group had a lower maximum creatine kinase–MB, a higher percentage of spontaneous defibrillation, a lower pulmonary capillary wedge pressure, and a lower requirement for dopamine than did the WB group, whereas the TC group failed to do better than the WB group.
Conclusions These results demonstrate that leukocyte-depleted reperfusion is potentially beneficial as an adjunct to terminal cardioplegia during cardiac surgery to attenuate reperfusion injury in patients with left ventricular hypertrophy.
Conventional methods of myocardial protection during open-heart surgery have generally provided satisfactory results for normal hearts.1 2 3 Nevertheless, intraoperative reperfusion injury in the myocardium can occur after a long period of AXC with the most up-to-date technique of myocardial protection in critical patients with LV hypertrophy.3 4 5 6 Myocardial tissue in the hypertrophied heart is more susceptible to ischemia/reperfusion injury than that in the normal heart, even under cardioplegic arrest.3 4 5 6 This is because the hypertrophied heart is characterized by energy depletion,7 morphological change,3 6 vulnerable calcium metabolism, and enhanced conversion of angiotensin I to angiotensin II contributing to diastolic dysfunction,8 all of which may decrease tolerance to AXC.
Several recent experimental studies have indicated the efficacy of neutrophil-depleted reperfusion because neutrophils and their products play a significant role in reperfusion injury and cause the no-reflow phenomenon.9 10 11 12 13 14 Hypertrophied heart appears to be more susceptible to neutrophil-mediated reperfusion injury. This speculation, however, has not been elucidated. In clinical situations, we applied leukocyte-depleted reperfusion as an adjunct to TC (LDTC) in patients undergoing CABG and proved its efficacy in terms of myocardial protection, particularly in patients with preoperative acute myocardial infarction.15 It was therefore thought that LDTC might also contribute to myocardial protection for patients with severe LV hypertrophy. However, no attempt has been made to apply this technique to patients with severe LV hypertrophy in view of the vulnerability of such hypertrophied heart to neutrophil-mediated reperfusion injury. This study was carried out to determine whether leukocyte-depleted reperfusion as an adjunct to terminal cardioplegia can attenuate reperfusion injury in the severe LV hypertrophied heart.
Thirty patients with severe LV hypertrophy who underwent cardiac surgery consisting of aortic valve replacement at Osaka University Hospital were enrolled in the study. All patients gave informed consent to participate in the study, and we followed the guidelines of our internal review board. None of the patients had coronary artery disease in addition to aortic disease. Their operations consisted of aortic valve replacement in 20 patients, aortic valve replacement combined with mitral valve replacement in 6 patients, and Bentall operation in 4 patients. The LV mass, as measured according to the method of Rackley et al,16 17 was in the range of >300 g (320 to 524 g), and LV end-systolic volume index, as measured according to the method of Wynne et al,17 18 was in the range of >100 mL/m2 (102 to 256 mL/m2). The age at surgery ranged from 30 to 71 years (mean, 52±14 years). All of the patients, the surgeon, the anesthesiologists, and the medical staffs were blinded as to which type of reperfusion was used. Patients were randomized into one of three groups for reperfusion: WB reperfusion (n=10), TC reperfusion (n=10), or LDTC reperfusion (n=10). No significant differences were noted among the three groups in age, AXC time, LV end-systolic volume index, LV mass, or preoperative cardiac functions (Table 1⇓).
The basic method of myocardial protection that we used was a cold potassium crystalloid cardioplegic solution (K+, 18 mEq/L, pH 7.8; osmolarity, 380 mOsm/mL; temperature, 4°C)5 15 that was delivered antegradely with direct coronary ostial cannulation. The initial dose for the cardioplegic solution was 3 mL/g LV mass, and a half-dose was reinfused every 30 minutes. Myocardial temperature was monitored at the LV apex and maintained at >15°C with topical cooling. The patients in the TC and the LDTC groups received 10 minutes of TC or LDTC, respectively, before the cessation of AXC.
Method of Controlled Reperfusion
The methods of controlled reperfusion used for the TC or LDTC group were performed as described previously.15 Briefly, arterial blood for TC and LDTC was circulated separately from the oxygenated reservoir after the completion of the surgical procedure and during AXC. Only for LDTC was a newly developed leukocyte-removal filter (Cellsorba-80P; Asahi Medical Co) incorporated just after the oxygenator reservoir to remove leukocytes. Then, arterial blood for the TC group or leukocyte-depleted arterial blood for the LDTC group was mixed with a cold potassium crystalloid cardioplegic solution with the use of double-head coupled roller pumps (Shiley Inc). The blood was then infused into the aorta through a cardioplegic cannula at a flow rate of 1 mL/min per gram of LV mass with a perfusion pressure of ≤60 mm Hg for 10 minutes. The temperature of blood was 30°C at the start of reperfusion and was increased immediately to 36°C. At the end of TC and LDTC reperfusions, the aorta was unclamped and the heart was reperfused with oxygenated whole blood through an aortic cannula in the same manner as for the WB group.
Leukocyte count and its differential count for neutrophils were obtained from the aortic root at three times: just before and during controlled reperfusion and immediately after the cardiopulmonary bypass.
In all patients, transmural biopsies from the LV apex were obtained with a Tru-Cut biopsy needle (Travenol) at Pre-Isc, at Post-Isc (≤1 minute after reperfusion), and at Rep.19 The site of the biopsies was the same as that used for ventricular venting during operation. The tissue samples were fixed immediately for 2 hours with a cold 3% glutaraldehyde solution and then prepared for electron microscopic examination as described previously.19 Ten sections from each block were taken randomly in each case at a magnification of ×1500 to ×10 000, and semiquantitative scoring was performed (range of 1 to 4, with the higher the score indicating the greater the damage) of subcellular changes such as mitochondrial damage, intracellular edema, nuclear change, and contraction band in the myocytes20 and in the capillary endothelial cells21 ; scoring was performed in a blinded fashion by two observers.
To count the number of neutrophils adhering to endothelial cells in the electron micrographs (×1500 to ×3000), visual identification of the nuclear shape in neutrophils and their direct contact with endothelial cells was used to distinguish them from the other leukocytes and/or neutrophils without adhesion. They were counted in 10 micrographs for each sample and described as counts per 100 capillaries.
MDA in the coronary sinus effluent blood and arterial blood was measured at 30 minutes of reperfusion with the use of high-pressure liquid chromatography and expressed in nanomoles per milliliter as described previously.12 The differences in MDA levels between coronary sinus effluent and arterial blood were compared among the three groups to obtain a value for myocardium-derived MDA.
During 24 postoperative hours, CI (L·min−1·m−2) and PCWP (mm Hg) were measured every 4 to 6 hours with a Swan-Ganz catheter in each patient. The mean values of CI and PCWP were compared among the three groups.
The rate of spontaneous defibrillation at the cessation of AXC, the dose of dopamine required for weaning of the cardiopulmonary bypass, and the length of the postoperative period on dopamine support were compared among the groups.
All of the results are expressed as mean±SD. An ANOVA with subsequent post hoc test for multiple comparisons was used to compare the data. For the percentage of spontaneous defibrillation, the χ2 test was used. A value of P<.05 was considered statistically significant.
With regard to operative death and survival, all patients tolerated the surgical procedures and survived without complications related to either TC or LDTC.
Before controlled reperfusion, no significant differences were found in the number of leukocytes (counts per cubic milliliter) or differential counts for neutrophils (%) among the WB (7300±900 counts/mm3, 67±13%), TC (8100±1000 counts/mm3, 61±14%), and LDTC (8500±1400 counts/mm3, 63±15%) groups. However, the LDTC group showed a significantly lower number of leukocytes and notably reduced differential counts for neutrophils in the aortic root (230±160 counts/mm3, 18±11%) than were seen in the TC (6600±1100 counts/mm3, 68±18%) and WB (7500±1500 counts/mm3, 67±16%, P<.01) groups. No significant difference was found in the number of leukocytes and differential counts for neutrophils immediately after cardiopulmonary bypass among the three groups (WB, 8900±2600, 68±13%; TC, 7400±1500, 65±18%; and LDTC, 7800±1300, 62±16%).
Findings of Representative Electron Micrographs
At 20 minutes of reperfusion in the WB and TC groups, wall thickening, damage to the nucleus, bleb formation, and neutrophils adhering tightly to the endothelial cells in the capillaries (Fig 1A⇓) were noted, accompanied by severely damaged myocytes in the WB group (Fig 1B⇓) and moderately damaged myocytes in the TC group (Fig 1C⇓). However, the LDTC group showed well-preserved myocardium (Fig 1D⇓).
Scores for Myocyte Changes
The scores at Pre-Isc, Post-Isc, and Rep were 0.3±0.1, 2.6±0.4, and 2.2±0.4 for the WB group; 0.4±0.1, 2.0±0.6, and 1.2±0.5 for the TC group; and 0.3±0.2, 2.3±0.4, and 0.6±0.3 for the LDTC group, respectively. Thus, there were no significant differences in Pre- and Post-Isc scores among the groups. However, the LDTC group showed a significantly lower Rep score than did the WB group (P<.05). Moreover, in the LDTC group, the Rep score was significantly lower than the Post-Isc score (P<.05), whereas no such differences were seen in the WB and TC groups (Fig 2⇓).
Scores for Capillary Endothelial Changes
The scores at Pre-Isc, Post-Isc, and Rep were 0.4±0.1, 1.8±0.3, and 1.9±0.2 for the WB group; 0.3±0.4, 1.8±0.6, and 1.6±0.4 for the TC group; and 0.2±0.2, 1.7±0.4, and 0.4±0.2 for the LDTC group, respectively. Again, there were no significant differences in Pre- and Post-Isc scores among the groups. However, the LDTC group showed a significantly lower Rep score than did either the WB or the TC group (P<.05). Only the LDTC group showed a significantly lower score at Rep than at Post-Isc (P<.05), whereas the WB and TC groups did not (Fig 3⇓).
Neutrophil Adherence in Capillaries
During the Post-Isc period, there was no significant difference in the number of neutrophils among the WB (0.2±0.2 counts/100 capillaries), TC (0.2±0.3), and LDTC (0.3±0.4) groups. During the Rep period, however, the LDTC group showed a significantly lower number of neutrophils (0.4±0.2) adhering to the endothelial cells than did either the WB (5.5±2.6) or TC (4.8±1.6) group (Fig 4⇓).
MDA Derived From Myocardium
The LDTC group showed a significantly lower count for MDA derived from the myocardium (0.15±0.11 nmol/mL) than did the WB (0.76±0.11) and TC (0.6±0.21, P<.05) groups (Fig 5a⇓).
The maximum values of CK-MB for 24 hours after surgery were 68±12 IU/L for the WB group, 49±16 IU/L for the TC group, and 21±13 IU/L for the LDTC group. The LDTC group thus showed significantly lower values than did the WB and TC groups, whereas there was no significant difference between the latter two groups (Fig 5b⇑).
Mean values of CI during 24 postoperative hours were 2.3±0.6 L·min−1·m−2 in the WB group, 2.4±0.8 in the TC group, and 2.8±0.6 in the LDTC group, respectively. There was no significant difference in CI among the groups (Fig 6⇓).
Mean values of PCWP during 24 postoperative hours were 15.3±2.6 mm Hg in the WB group, 12.4±4.8 in the TC group, and 9.8±4.6 in the LDTC group. The LDTC group showed a significantly lower value than did the WB group. There were no significant differences in the values of PCWP between the WB and TC groups and between the TC and LDTC groups (Fig 6⇑).
Other Clinical Results
The LDTC group showed a significantly higher percentage of patients with spontaneous defibrillation after the release of AXC, less dopamine required for weaning of the cardiopulmonary bypass, and a shorter postoperative period on dopamine support than the WB group, whereas there were no significant differences in these data between the TC and LDTC groups or the TC and WB groups (Table 2⇓).
The results of this clinical study have clearly demonstrated that LDTC significantly reduced the degree of myocyte and capillary endothelial damage in patients with LV hypertrophy, whereas TC alone did not attenuate such endothelial cell damage. Furthermore, LDTC resulted in a significantly lower number of neutrophils adhering to the endothelial cells, followed by less production of MDA derived from the myocardium compared with results for the TC and WB groups. Consequently, patients who had received LDTC showed a greater improvement in their clinical parameters, such as less leakage of CK-MB, a higher percentage of spontaneous defibrillation, and lower PCWP during the first 24 postoperative hours, with a lower requirement for dopamine support than even those in the WB group, whereas patients with TC alone did not show any improvement in these clinical data. These results demonstrate that leukocyte depletion attenuates reperfusion injury in patients with LV hypertrophy and is thus potentially beneficial as an adjunct to terminal cardioplegia during open-heart surgery.
The earliest direct evidence suggesting the involvement of leukocytes in myocardial reperfusion injury was the capillary plugging by leukocytes after myocardial ischemia and reperfusion in dogs reported by Engler and colleagues.10 They were also the first to determine the positive effect of leukocyte depletion on the no-reflow phenomenon in canine hearts subjected to ischemia/reperfusion.22 Several subsequent studies have reported the efficacy of leukocyte-removal filters in attenuation of reperfusion injury.23 Neutropenia induced by a leukocyte-removal filter was found to reduce microvascular permeability, suggesting that neutrophils are largely responsible for coronary endothelial dysfunction.24 These experimental studies are consistent with the results of our clinical study. Undoubtedly, therefore, leukocyte-depleted reperfusion plays a significant role in attenuation of reperfusion injury in the human heart.
However, it remains controversial whether neutrophils are involved in the stunned myocardium.25 In our previous study,15 the results of the clinical application of LDTC in elective CABG, which can cause relatively mild reperfusion injury resembling that caused by the stunned myocardium, did not differ significantly from those for the WB group or the TC group. This result is consistent with past reports based on animal models that found no involvement of neutrophils in the mechanisms of the stunned myocardium.25 It is therefore reasonable to assume that the role of neutrophils in reperfusion injury depends on the degree of myocardial damage, thus suggesting the limiting effect of leukocyte depletion on the stunned myocardium. However, the results of both emergency CABG reported previously15 and patients undergoing aortic valve replacement with LV hypertrophy in this study show significant differences between the LDTC group and either the WB or TC group. This suggests that removal of leukocytes is useful when serious reperfusion injury is expected to occur during open-heart surgery, especially in compromised hearts.
Myocardial tissue in the hypertrophied heart is more susceptible to ischemia/reperfusion injury than that in the normal heart, even during cardioplegic arrest.3 4 5 6 However, so far no reports have elucidated the role of neutrophils in this susceptibility. The data presented in the present study appear to provide direct evidence that the superior recovery of the LDTC group may be related to the depletion of leukocytes. This leads us to speculate that the hypertrophied heart may be susceptible to neutrophil-mediated reperfusion injury even though we did not perform a comparison of such susceptibility between normal and hypertrophied hearts. Although this mechanism has never been reported, it may be speculated that neutrophils have a greater effect on the hypertrophied heart because subsequent ischemic damage is more severe in the hypertrophied than in the normal heart. Therefore, neutrophils appear to exacerbate reperfusion injury in the hypertrophied heart.
Recent reports have shown that endothelial cells may play a key role in the evolution of neutrophil-induced reperfusion injury.13 In this study, ultrastructural assessment proved that reperfusion with whole blood, even during terminal blood cardioplegia, caused a significantly larger number of neutrophils to adhere to the endothelial cells accompanied by more subcellular alterations in the hypertrophied heart than did the reperfusion with leukocyte-depleted blood. This emphasizes again the importance of endothelial protection to attenuate reperfusion injury, particularly in patients with compromised hearts such as those with LV hypertrophy.13
During surgery, activation of neutrophils can be easily evoked by complement activation during open-heart surgery.26 When activated, neutrophils produce superoxide anions by means of NADPH oxidase. Furthermore, they release myeloperoxidase, which catalyzes the production of OCl−, a greater oxygen radical. Neutrophils also produce nitric oxide, which can react with superoxide anions to yield ONOO−, one of the most active radicals.27 Therefore, activation of neutrophils produces various oxygen radicals, resulting in the exacerbation of reperfusion injury, especially during open-heart surgery. This suggests the importance of leukocyte-depleted reperfusion during open-heart surgery, although no clinical application, except our method, has been reported, in part because of the technical difficulties, the lack of a suitable filter for coronary circulation with heparinized blood, or both.15
The principal advantage of LDTC lies in the combination of TC and reperfusion with leukocyte-depleted blood because of its technical convenience and improvement in the existing methods of myocardial protection.15 To attenuate reperfusion injury in the myocardium, terminal blood cardioplegia has been proposed and used in clinical situations.28 29 30 TC has also been reported to accelerate myocardial metabolic recovery and preserve high-energy phosphates.28 29 30 This effect may contribute to attenuation of the damage, mainly in myocytes, because the procedure failed to prevent neutrophil adherence to endothelial cells in our study. On the other hand, leukocyte-depleted reperfusion alone reportedly makes other contributions, such as the attenuation of damage to endothelial cells mainly caused by neutrophil adherence to such cells.15 Because the efficacy of leukocyte-depleted reperfusion alone was not examined in the present study, the precise role of leukocyte depletion alone in the human heart with LV hypertrophy could not be clarified. However, our results suggest that the combination of TC and leukocyte-depleted reperfusion in LDTC appears to provide better preservation of the human myocardium with LV hypertrophy than does either of these procedures alone. As for the pathogenesis of reperfusion injury in myocardium, a number of factors have been proposed as an explanation.31 Therefore, to improve myocardial protection, particularly for patients with compromised hearts, a combination of several myocardial protective methods with several different mechanisms might be required, and LDTC appears to be a good candidate for this strategy.
Our method for reperfusion with leukocyte-depleted blood as an adjunct to TC consisted of 10 minutes of controlled reperfusion with leukocyte-depleted blood for only the coronary circulation. These first 10 minutes of reperfusion are the most critical period for the morphological changes at subcellular levels, such as neutrophil sequestration, resulting in the no-reflow phenomenon.31 Ultrastructural assessment has proved to be extremely useful for determination of the efficacy of myocardial protection.18 19 20 21 31 For this reason, we performed LV biopsy and evaluated the subcellular alterations to compare the extent of myocardial protection in each group. Our results indicated that 10 minutes of LDTC appears to be sufficient, even for patients with LV hypertrophy, to attenuate neutrophil-mediated reperfusion injury, as has been proposed previously.32 33 Further studies are essential for the enhancement of myocardial protection as one of the future perspectives.
In summary, LDTC was used for a series of patients with LV hypertrophy. The myocardial biopsy obtained at reperfusion showed better mitochondrial preservation in myocytes and capillary endothelial preservation, with fewer neutrophils adhering to endothelial cells for the LDTC group. These improvements were accompanied by enhanced clinical parameters. Our results demonstrate that leukocyte-depleted reperfusion attenuates reperfusion injury in patients with LV hypertrophy and thus is potentially beneficial as an adjunct to TC during cardiac surgery.
Selected Abbreviations and Acronyms
|CABG||=||coronary artery bypass graft surgery|
|LDTC||=||leukocyte-depleted terminal cardioplegia|
|PCWP||=||pulmonary capillary wedge pressure|
|Rep||=||15 minutes after reperfusion|
Reprint requests to Hikaru Matsuda, MD, First Department of Surgery, Osaka University Medical School, 2-2 Yamada-oka, Suita, Osaka 565, Japan.
- Received September 13, 1995.
- Revision received October 23, 1995.
- Accepted November 9, 1995.
- Copyright © 1996 by American Heart Association
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