Ort h otopic heart transplantation has been demonstrated

Transcription

1 Study of Efficacies of Leukocyte-Depleted Terminal Blood Cardioplegia in 24-Hour Preserved Hearts Norihide Fukushima, MD, Ryota Shirakura, MD, Seizou Nakata, MD, Mitsunori Kaneko, MD, Shuji Miyagawa, MD, Yoshifumi Naka, MD, [ou-chen Chang, MD, Goro Matsumiya, MD, Susumu Nakano, MD, and Hikaru Matsuda, MD First Department of Surgery, Osaka University Medical School, Osaka, Japan To evaluate the effect of leukocyte-depleted terminal blood cardioplegia on prolonged preservation, 41 canine hearts were stored in modified Collins' solution and transplanted heterotopically. Hearts were transplanted soon after harvesting in group 1 and after 24-hour preservation in groups 2, 3, and 4. Blood cardioplegia was applied just before aortic undamping in groups 3 and 4; group 3 received simple blood cardioplegia and group 4 received leukocyte-depleted cardioplegia. The percentage of the preload recruitable stroke work and diastolic compliance after transplantation compared with the preharvesting value in group 4 did not differ from those in group I, but the percentage of the preload recruitable stroke work in groups 2 and 3 was significantly lower than that in groups 1 and 4. The percentage of diastolic compliance in groups 2 and 3 was significantly higher than that in groups 1 and 4. Coronary blood flow 40 minutes after aortic undamping in group 4 did not differ from that in group I, but was significantly higher than the blood flows in groups 2 and 3. Significant production of malondialdehyde was detected during terminal blood cardioplegia and 10 minutes after aortic undamping in groups 2 and 3, but never in groups 1 and 4. After leukocyte-depleted terminal cardioplegia, the myocardial adenosine triphosphate content increased to the preharvesting value in group 4. Our results suggest that leukocyte-depleted terminal blood cardioplegia may be effective in replenishing the energy-depleted myocardium and reducing reperfusion injury, resulting in adequate cardiac function. (Ann Thome Surg ) Ort h otopic heart transplantation has been demonstrated to be an established therapy for patients in an otherwise unsalvageable state because of end-stage heart disease; however, the current supply of donor hearts is unfortunately severely limited. Moreover, ventricular failure, probably stemming from inadequate preservation, is the leading cause of death early after transplantation [1]. To extend the donor pool as well as to reduce the graft failure rate, it is necessary to improve the methods for prolonged preservation. As one of the strategies for this, leukocyte-depleted terminal blood cardioplegia (LDTC) may be useful. Using heterotopic and orthotopic heart transplantation models, we have shown that LDTC leads to adequate cardiac function in heart grafts preserved for 24 hours [2]. In the present study, we studied the effects of LDTC on coronary vessels, oxygen free-radical production, and the myocardial adenosine triphosphate (ATP) content using the heterotopic heart transplantation model to elucidate the mechanism of LDTC responsible for prolonged heart preservation. Accepted for publication May 9, Address reprint requests to Dr Fukushima, First Department of Surgery, Osaka University Medical School, 2-2 Yamada-Oka, Suita, Osaka 565, Japan. 1994by The Society of Thoracic Surgeons Material and Methods Material Donor hearts were obtained from 41 mongrel dogs of either sex weighing 5.6 to 9.6 kg (average, 7.4 ::': 2.3 kg) and transplanted heterotopically in the retroperitoneal space of hosts weighing 10.1 to 19.6 kg (average, 14.5 ::': 2.3 kg). Animal care conformed to the standards of the National Society for Medical Research (Principles of Laboratory Animal Care) and the "Guide for the Care and Use of Laboratory Animals" (NIH publication 85-23, revised 1985). Four groups were compared. Group 1 hearts (n = 8) were transplanted soon after harvesting. Hearts in groups 2 (n = 12),3 (n = 9), and 4 (n = 12) were preserved in modified Collins' solution for 24 hours. Groups 1 and 2 grafts were reperfused without any intervention. Group 3 grafts were reperfused with simple warm blood cardioplegia (TC) and LDTC was used in group 4 grafts. Haroesting and Preseroaiion The dogs were placed on a ventilator, and a bilateral horizontal thoracotomy through the fourth intercostal space was made. After systemic heparinization, the heart was decompressed by opening the inferior vena cava. The aortic arch was cross-clamped between the brachiocephalic and left subclavian artery, and cold potassiumenriched crystalloid cardioplegic solution was adminis /94/$7.00

2 1652 FUKUSHIMA ET AL Ann Thorac Surg tered through the catheter in the brachiocephalic artery. The heart was then excised rapidly, dividing and ligating the superior and inferior venae cavae, the azygos vein, and the pulmonary veins. The coronary vascular bed was washed out with modified Collins' solution (4 mllkg) to replace the cardioplegic solution, using the method of Kohno and colleagues [3]. The heart was immersed in modified Collins' solution containing 3 meq/l of magnesium, 0.5 mg/l of verapamil, and 0.6 ng/l of prostacyclin analogue (OP-41483, PGI 2 ). The bath was maintained at 4 :': 1 C in a refrigerated room. Method of Transplantation The donor heart was transplanted heterotopically in an intact dog, as previously described [2]. The donor aorta was anastomosed end-to-side with the recipient abdominal aorta. After the aortic anastomosis was created, group 3 grafts were perfused for 10 minutes with simple TC and group 4 grafts with LDTC before aortic unclamping (AVC). After AVC, coronary sinus blood from the graft was drained into the host circulation through a bypass between the donor right ventricle and the recipient superior vena cava. The warm ischemic time during transplantation was 11.2 :': 1.7 minutes. Terminal Blood Cardioplegia Blood cardioplegia containing 20 meq/l of potassium, with the hematocrit adjusted to about 20% and at a temperature of 26 C, was used. The grafts were perfused antegradely with TC at a pressure of 60 em H 2 0 for 10 minutes before AVC in groups 3 and 4. In group 4, leukocytes were removed from the TC by a polyester filter (Sepacell 500A; Asahi Medical, Tokyo, Japan) inserted into the cardioplegia delivery catheter. This reduced the white blood cell count in the blood cardioplegic solution from 4,400 :': 750/mm 3 to 72 :': 24/mm 3 Neutrophils were almost absent when examined for by usual manual microscopy. The platelet count decreased from 15.2 :': 1.2/mm 3 to 4.5 :': 0.9/mm 3 Cardiac Function Measurement Preload recruitable stroke work and diastolic compliance were measured before harvesting (baseline value) and 40 minutes after AVe. A SF micromanometer (Model MPC 500; Millar Instruments, Houston, TX) and an 8F conductance catheter (Cordis Europa, Roden, The Netherlands) were inserted into the left ventricle through the apex to allow the left ventricular pressure and volume, respectively, to be measured (Fig 1). The left ventricular pressure-volume loop was obtained as previously shown [2, 4]. At each sampling point, stroke work (SW) and enddiastolic pressure (EDP) and volume (EDV) of six to eight cardiac cycles were measured. By linear regression analysis, data were fitted to the following formula: SW = MI (EDV - VI) EDP = M 2 (EDV - V0, where the slopes M 1 and M 2 were the preload recruitable stroke work and diastolic compliance, respectively. r"",r.-...y1lonn:",."nnnnrlfr,1 magnetic flow meter I ;=jcoronary return arterial blood conductance catheter Fig 1. Experimental model. (LA = left atrium; LV = left ventricle; PA = pulmonary artery; RA = right atrium; RV = right ventricle; SVC = superior vena cava.) Myocardial Adenosine Triphosphate Content Full-thickness transmyocardial left ventricular samples for determination of the myocardial ATP content were obtained with a high-speed biopsy drill at harvesting, after 24 hours of preservation, and at 10 and 40 minutes after AVe. Samples were assayed by high-pressure liquid chromatography (C-9A system; Shimazu, Tokyo, Japan) with a standard solution of ATP, as described by Kamiike and associates [5]. Coronary Blood Flow During perfusion with TC, coronary venous return was measured with a timed collection in groups 3 and 4. After AVC, coronary venous return from the graft was drained into the host circulation through the bypass between the right ventricle of the graft and the superior vena cava of the host. The flow of the bypass, which was identified to be the coronary blood flow (CBF) of the grafts, was measured continuously by an electromagnetic blood flowmeter (MFV-1200; Nihon-Koden, Tokyo, Japan) (see Fig 1). Production of Malondialdehyde After 5 and 10 minutes of TC, and at 5, 10, and 20 minutes after AVC, aortic and coronary sinus blood samples were obtained and the malondialdehyde (MDA) content measured (in nanomoles per milliliter) with a fluorescence assay by Shionogi biomedical laboratory. The production of MDA (measured in nanomoles per minute) was calculated by the formula: MDA production = (MDA of CS - MDA of Ao) x CBF, where CS is coronary sinus blood and Ao is aortic blood. Myocardial Water Content Forty minutes after AVC, the grafts were excised. Left ventricular free walls and ventricular septa of the grafts

3 Ann Thorac Surg FUKUSHIMA ET AL 1653 were taken and weighed, then desiccated at 100 C for 48 hours and reweighed. The myocardial water content (MWC) was calculated by the formula: dry Weight) MWC = 1-. X 100(%) ( wet weight Statistical Analysis Statistical analysis was performed using the multiple comparison analysis (analysis of variance). Results are reported as the mean::+: a standard deviation. A p value of less than 0.05 was considered significant. 'Results Posttransplantation Cardiac Function The rates of spontaneous beating in groups 1, 2, 3, and 4 were 75% (6/8), 0% (0/12), 33% (3/9), and 100% (12112), respectively. Those in groups 2 and 3 were significantly lower than those in groups 1 and 4 (p < 0.01 and < 0.05, respectively) (Table 1). The percentage of the posttransplantation preload recruitable stroke work compared to the baseline value in group 4 (140% ::+: 11%) did not differ from that in group 1 (120% ::+: 11%). Those in group 2 (51% ::+: 4%; P < 0.01) and group 3 (83% ::+: 10%; P < 0.05) were significantly lower than those in groups 1 and 4. The percentage of posttransplantation diastolic compliance compared with the baseline value in group 4 (99% ::+: 4%) was not different from that in group 1 (98% ::+: 3%). Those in group 2 (1,490% ::+: 470%; P < 0.01) and group 3 (570% ::+: 230%; P < 0.01) were significantly higher than those in groups 1 and 4. Myocardial Adenosine Triphosphate Content The myocardial ATP content in groups 2, 3, and 4 decreased significantly to 14.1 ::+: 1.2, 14.2 ::+: 1.7, and 13.6 ::+: 1.5 /Lmollg dry weight, respectively, after 24 hours of immersed preservation, and those in groups 3 and 4 significantly increased after TC. The content at the end of TC in group 3 was significantly lower than the preharvesting value (21.3 ::+: 1.4 versus 23.8 ::+: 1.3 /Lmollg dry Table 1. Comparison of the Cardiac Function Variables After Heterotopic Transplantation" Variable Spontaneous beating Preload recruitable stroke work index (%)d Diastolic compliance (%)d Group 1 6/8 (75%) 120 ± ± 3 Group 2 Group 3 Group 4 0/12 b 3/9 (33%)C 12/12 (100%) 83 ± 10 c 1,490 ± 470 b 570 ± 230 b 140 ± ± 4 a See text for explanation of groups. Data are shown as the mean :': a standard deviation. b p < c P < 0.05 versus groups 1 and 4. d Percentage compared with the preharvesting value. (tlmol/dry weight) 30, '------'----= ' :-- immersed storage (1.25 I- :)0 «15 «10 ::E 5 - -<) - GROUP-1 (on-site Tx) CJ GROUP-2(24 hrs storage) GROUP-3 (24 hrs + simple Te) ---e-- GROUP4(24 hrs +LDTC) TIME(hrs) *: p<0.05 vs GROUP (min) Fig 2. Changes in myocardial adenosine triphosphate (ATP) content during storage, during terminal cardioplegia, and 40 minutes after aortic unclamping. Data are shown as the mean ± a standard deviation. (LDTC = leukocyte-depleted terminal blood cardioplegia; TC = terminal blood cardioplegia; Tx = heart transplantation.) weight; p < 0.05), but that in group 4 at the end of LDTC was as high as the preharvesting value (24.1 ::+: 1.7 versus 23.5 ::+: 1.5 /Lmollg dry weight). Forty minutes after AVC, the myocardial ATP content in groups 1, 3, and 4 (24.1 ::+: 1.2, 24.1 ::+: 1.2, and 24.2 ::+: 1.4 /Lmollg dry weight, respectively) were not significantly different from the preharvesting value. However, the ATP content in group 2 40 minutes after AVC was significantly lower than the preharvesting value (20.2 ::+: 1.5 versus 23.1 ::+: 1.3 /Lmollg dry weight; p < 0.05) and those in the other groups (p < 0.05) (Fig 2). Coronary Blood Flow Ten minutes after TC, the CBF in group 4 (45 ::+: 7 mllmin) was not different from that in group 3 (55 ::+: 8 mllmin). Five minutes after AVC, the CBF in group 4 (40 ::+: 7 mllmin) was significantly lower than those in group 1 (72 ::+: 7 mllmin) and group 2 (67 ::+: 9 mllmin) (p < 0.05). At 20 and 40 minutes after AVC, the CBFs in group 4 (38 ::+: 6 and 39 ::+: 4 mllmin, respectively) were not significantly different from those in group 1 (40 ::+: 5 and 40 ::+: 4 mllmin, respectively) but was significantly higher than those in group 2 (24 ::+: 5 and 25 ::+: 5 mllmin, respectively; both p < 0.05) and group 3 (26 ::+: 5 and 25 ::+: 5 mllmin, respectively; both p < 0.05) (Fig 3). Production ofmalondialdehyde After 10 minutes of TC, MDA production in group 4 (-17.1 ::+: 6.8 nmollmin) was significantly lower than that in group 3 (49.7 ::+: 13 nmollmin). Ten minutes after AVC, MDA production in group 4 (-1.1 ::+: 4.2 nmollmin) did not differ from that in group 1 (-6.6 ::+: 6.2 nmollmin), but was significantly lower than those in groups 2 and 3 (11.5 ::+: 4.9 and 30.1 ::+: 15 nmollmin, respectively; p < 0.05) (Fig 4). Myocardial Water Content The myocardial water content 40 minutes after AVC in group 4 (85.6% ::+: 0.6%) did not differ from that in group

4 1654 FUKUSHIMA ET AL Ann Thorac Surg (ml/m...in): , 80, _.-0-- GROUP-1 (on-ssetx) o GROUP-2 (24hrs storage) GROUP-3 (24hrs+ simple TC) GROUP-4 (24hrs+ LOTC) o 9 CJ)40 -c 20 a: o U o ')\'. t **. i"'\ l"/l \- i......:., * : P<0.05 vs GROUP-1 and -4 **: P<0.05 vs GROUP ro TIME after aortic unclamping (min) Fig 3. Changes in coronary blood flow during terminal cardioplegia and for 40 minutes after aortic unclamping. Data are shown as the mean ::':: a standard deviation. (LDTC = leukocyte-depleted terminal blood cardioplegia; TC = terminal blood cardioplegia; Tx = heart transplantation.) ('*0> 100 -c Q)-C 0 o Cl) as... a; i3 80 rj 0>- ::::E Group-1 (_Tx) Fig 5. Myocardial water content ofthe grafts 40 minutes after aortic unclamping. Data are shown as the mean ::':: a standard deviation. (LDTC = leukocyte-depleted terminal blood cardioplegia; NS = not significant; TC = terminal blood cardioplegia; Tx = heart transplantation.) 1 (84.8% ± 1.1%) but was significantly lower than those in groups 2 and 3 (88.4% ± 0.6% and 88.0% ± 0.8%, respectively; p < 0.05) (Fig 5). Comment Simple hypothermic storage and continuous hypothermic perfusion have been the two main methods of heart preservation for transplantation. Simple hypothermic storage is easy to handle and makes distant procurement easier. Findings from previous experimental studies have shown that a storage solution consisting of intracellular electrolyte components appears to prolong the period of simple immersion [6-8]. However, modification of the storage solution alone could not prolong the safe preservation period to as long as 24 hours [7,8]. As one of the strategies to reduce the ischemic myo- (nmol/min) 6O"':""" ';=""""''-=::-::-;;:=T:' ' -*"l-. GROUP-1 (on-snetx) 40 Z Z Q 20 b :::> Cl a: a, r I during Te [J... GROUP-2 (24 hrs storage)... GROUP-3 (24 hrs + simple TC) "'. =-=GROUP-4 (24 hrs + LDTC)... * * T""'L'..."."..1 I] 't] o 0...=.. {9' : _.. J.:;====='II'1 -t-- I *: P<O.05 vs GROUP-1 and after aortic unclamping (min) TIME Fig 4. Changes in production ofmalondialdehyde (MDA) during terminal blood cardioplegia and for 20 minutes after aortic unclamping. Data are shown as the mean ::':: a standard deviation. (LDTC = leukocyte-depleted terminal blood cardioplegia; TC = terminal blood cardioplegia; Tx = heart transplantation.) cardial injuries after preservation, myocardial energy store replenishment and attenuation of reperfusion injury may have a role. This is particularly important when the preservation period is prolonged. Swanson [9] and Milliken [10] and their associates examined the effect of TC on hearts preserved for 24 hours to evaluate the effect of myocardial energy store replenishment. Breda [11] and Mollhoff [12] and their associates examined the attenuation of reperfusion with leukocyte-depleted blood, and showed that a single intervention for replenishment of energy-depleted myocardium or for prevention of myocardial injury at reperfusion was insufficient to extend the safe ischemic period to as much as 24 hours. We have previously shown that LDTC provided adequate systemic cardiac function to heart grafts preserved for 24 hours [2]. In the present study, we also examined the spontaneous beating rate and diastolic compliance. Beating was restored spontaneously in all grafts in group 4 (the 24-hour LDTC group), but this occurred in no grafts in group 2 (the simple TC group) and in only 3 of 9 grafts in group 3 (the 24-hour TC group). The posttransplantation diastolic compliance in group 4 was not different from the preharvesting value (baseline) or the posttransplantation value in group 1 (the on-site transplantation group), but it was significantly lower than the posttransplantation values in groups 2 and 3. These data suggest that LDTC may preserve diastolic function and conduction as well as systemic function after 24-hour storage. It seems unlikely that only 10 minutes of LDTC would be enough to reverse the injury to the heart caused by 24-hour storage. However, as we previously reported [2], the cardiac function findings in orthotopically transplanted grafts in a 24-hour LDTC group were comparable to the preharvesting and posttransplantation results in the on-site transplantation group, but no dog in the simple 24-hour storage group could be weaned from cardiopulmonary bypass, even with inotropic drug sup-

5 Ann Thorae Surg FUKUSHIMA ET AL 1655 port. Moreover, the dogs in the 24-hour LDTC group showed cardiac function at least 6 hours after orthotopic heart transplantation that was comparable to that in the on-site transplantation group (unpublished data). These data suggest that even only 10 minutes of LDTC can modify or reduce the injury caused by 24-hour storage as well as that occurring after reperfusion. Lazar and associates have shown that a major metabolic deficit caused by ischemia in myocardium is a limited capacity to utilize delivered oxygen [13], and that secondary blood cardioplegia plays an active role in the reversal of ischemically damaged myocardium [14]. Takami and associates [15] have shown that myocardial ischemic damage becomes irreversible if the myocardial ATP content decreases below 40% of the pre-preservation value. In the present study, after 24 hours of immersed preservation in our modified Collins' solution, the myocardial ATP content decreased to 54% of the pre-preservation value, which remained in the reversible range. Therefore, TC and LDTC may play a role in reversing the myocardial damage caused by prolonged preservation. The myocardial ATP content returned to the pre-preservation value just after LDTC in group 4 and 40 minutes after AVC in group 3, but, in group 2, only up to 82% of the pre-preservation value was observed 40 minutes after AVe. The improvement in oxidative metabolism brought about by LDTC or TC was reflected in the final measurements of ventricular performance. On reperfusion, neutrophils promptly infiltrate into ischemically damaged tissue [16], but the mechanism by which leukocytes cause reperfusion injury is unclear. Studies conducted by Engler and associates [17] have shown that neutrophils, in part because of their large size and relative lack of deformability, plug myocardial capillaries during reperfusion, and that these cells appear to actively adhere to the endothelium of formerly ischemic myocapillaries. This finding may underlie the no-reflow phenomenon seen after ischemia and reperfusion. In the present study, there was no difference in CBF during TC, with or without leukocyte depletion. In groups 1 and 2, a transient burst of CBF was observed soon after reperfusion, followed by a subsequent decrease in CBF, but no such burst or subsequent decrease was observed in group 4. The CBF 40 minutes after AVC in group 4 did not differ from that in group 1, but was significantly higher than the CBFs in groups 2 and 3. These data suggest that leukocytes are activated and playa role in the no-reflew phenomenon arising immediately after reperfusion with normal arterial blood. Another mechanism by which leukocytes cause reperfusion injury correlates with their release of oxygen free radicals [18-20]. The oxygen free radicals directly damage the endothelium of capillaries and the myocardium, and also generate a potent chemotactic factor for neutrophils, thereby creating a positive feedback cycle for the generation of more oxygen free radicals [19]. Kim and associates [20] have shown that MDA, a by-product of free radical-mediated lipid peroxidation, is released during reperfusion. In the present study, a significant pro- duction of MDA was detected during simple TC and after AVC in group 3, but no production of MDA was detected during LDTC and after AVC in group 4. These data support the hypothesis that one of the principal sources of oxygen free radicals during reperfusion is leukocytes in reperfusate. In conclusion, the reduction in the myocardial ATP content during 24-hour hypothermic immersed preservation was fully replenished by LDTe. Leukocytedepleted terminal blood cardioplegia maintained coronary perfusion after AVC and prevented the production of oxygen free radicals during TC and after AVe. These improvements might provide good cardiac function in grafts after 24-hour storage comparable to that observed in on-site transplanted hearts. Our results suggest that LDTC would extend the safe preservation period for heart transplants. We gratefully acknowledge the generous provision of prostacydin analogue OP (PGI 2A) by Ono Pharmacological Company, Osaka, Japan. References 1. Kriett JM, Kaye MP. The registry of the International Society for Heart and Lung Transplantation: eighth official report J Heart Lung Transplant 1991;10: Fukushima N, Shirakura R, Nakata S, et a1. Effects of terminal cardioplegia with leukocyte-depleted blood on heart grafts preserved for 24 hours. J Heart Lung Transplant 1992;11: Kohno H, Shiki K, Ueno Y, Tokunaga K. Cold storage of the rat heart for transplantation. Two types of solution required for optimal preservation. J Thorac Cardiovasc Surg 1987;93: Shirakura R, Matsuda H, Nakano S, et a1. Cardiac function and myocardial performance of 24-hour-preserved asphyxiated canine hearts. Ann Thorac Surg 1992;53: Kamiike W, Watanabe F, Hashimoto T, Tagawa K, Nakao K, Kawashima Y. Changes of cellular levels of ATP and its catabolites in ischemic rat liver. J Biochem 1982;91: Lower RR, Shumway NE. Studies on orthotopic transplantation of the canine heart. Surg Forum 1960;11: Reitz BA, Brody WR, Hickey PR, Michaelis LL. Protection of the heart for 24h with intracellular (high K) solution and hypothermia. Surg Forum 1974;25: Thomas FT, Schatzki PF, Hudson BH, Wolf JS. Successful 24-hr ischemic cardiac preservation using a new hyperosmolar perfusate. Surg Forum 1975;26: Swanson DK, Dufek JH, Barber TA, Kahn DR. Improving function of hearts preserved for 24 hours by controlling reperfusion. Transplantation 1979;28: Milliken JC, Billingsley AM, Laks H. Modified reperfusate after long-term preservation of the heart. Ann Thorac Surg 1989;47: Breda MA, Drinkwater DC, Laks H. Prevention of reperfusion injury in the neonatal heart with leukocyte-depleted blood. J Thorac Cardiovasc Surg 1989;97: Mollhoff T, Sukehiro S, Van Aken H, Flameng W. Reperfusion injury after 24 hours cold storage of donor hearts. Possible role of leukocyte activation. Thorac Cardiovasc Surg 1990;38: Lazar HL, Buckberg GD, Manganaro AJ, Becker H. Reversal of ischemic damage with secondary blood cardioplegia. J Thorac Cardiovasc Surg 1979;78: Lazar HL, Buckberg GD, Manganaro AM, Becker H. Myocardial energy replenishment and reversal of ischemic dam-

6 1656 FUKUSHIMA ET AL Ann Thorae Surg age by substrate enhancement of secondary blood cardioplegia with amino acid reperfusion. J Thorac Cardiovasc Surg 1980;80: Takami H, Furuya E, Tagawa K, et al. NMR-invisible ATP in rat hearts and its change in ischemia. J Biochem (Tokyo) 1988;104: Chatelain P, Latour JG, Tran D, de Lorgeril M, Dupras G, Bourassa M. Neutrophil accumulation in experimental myocardial infarcts: relation with extent of injury and effect of reperfusion. Circulation 1987;75: Engler RL, Schmid-Schonbein GW, Pavelec RS. Leukocyte capillary plugging in myocardial ischemia and reperfusing in the dog. Am J Pathol 1983;111: Mahta JL, Nichols WW, Mehta P. Neutrophils as potential participants in acute myocardial ischemia: relevance to reperfusion. JAm Coll Cardiol 1988;11: Hammond B, Hess ML. The oxygen free radical system: potential mediator of myocardial injury. J Am Coll Cardiol 1985;6: Kim M, Akera T. O 2 free radicals: cause of ischemiareperfusion injury to cardiac Na+-K+-ATPase. Am J Physiol 1987;252:H INVITED COMMENTARY This article by Fukushima and associates represents a continuation of a study that they published in the Journal of Heart and Lung Transplantation in 1992, in which they showed that terminal blood cardioplegia that was leukocyte depleted improved graft survival in 24-hour preserved hearts. The present study expands on this initial report by looking at several new factors, including the production of malondialdehyde as an index of free radical formation, coronary blood flow as an index of the no-reflew phenomenon, and the adenosine triphosphate content in various experimental groups. In addition, they looked at the preload recruitable stroke work and diastolic compliance as load-independent measures of ventricular function. In this 24-hour model of ischemia and reperfusion, the adenosine triphosphate content is maintained when terminal blood cardioplegia with leukocyte depletion is used. Changes in coronary blood flow as an index of no reflow and changes in the production of malondialehyde as an index of free radical formation are not detected. We now recognize that the end result of ischemia and reperfusion is a summation of factors that are contributed to by both cellular and noncellular (humoral) mechanisms and compounded by changes induced in the target organ itself by the ischemic event. The interplay between these various contributors to the end result (organ dysfunction) is complex and interdependent. Thus, changes in the endothelium brought about by ischemia increase leukocyte accumulation in the target organ, which provides a concentrated source for free radical formation and the local release of proteolytic enzymes. Activation of the complement cascade, along with leukotriene and cytokine release, can enhance local damage in concert with the leukocytes or through more direct, noncellular mechanisms. Closely correlated are changes in prostanoid metabolism and the release of vasoactive peptides. As intertwined as this sequence of events is, the removal of key protagonists can reduce the extent of organ damage or dysfunction and lessen the effect of those factors still present. Reducing the deleterious effect of leukocytes by physical or chemical means falls into this category. The article by Fukushima and colleagues, therefore, demonstrates that, in the setting of global 24-hour ischemia followed by reperfusion, removal of leukocytes during reperfusion significantly improves recovery, although many of the noncellular mechanisms that can lead to organ dysfunction are still fully intact. With this work of Fukushima and his colleagues and support for this concept in the literature, the use of leukocyte-depleted blood during reperfusion is worthy of well-controlled, randomized clinical trials. Bartley P. Griffith, MD Brack G. Hattler, MD, PhD Division of Cardiothoracic Surgery University of Pittsburgh School of Medicine C700 Presbyterian University Hospital 200 Lothrop St Pittsburgh, PA 15213