Role of leukocyte depletion during cardiopulmonary bypass and cardioplegic arrest

Role of Leukocyte Depletion During Cardiopulmonary Bypass and Cardioplegic Arrest Harold L. Lazar, MD, Xi Zhang, MD, PhD, Takafumi Hamasaki, MD, Patrick Treanor, CCP, Samuel Rivers, BS, Sheilah Bernard, MD, and Richard J. Shemin, MD Department of Cardiothoracic Surgery, The Boston University Medical Center, Boston, Massachusetts

Background. Leukocyte depletion (LD) has been shown to be beneficial during the reperfusion of acutely ischemic myocardium; however, its role during cardiopulmonary bypass (CPB) in hearts protected with blood cardioplegia (BCP) is u n k n o w n . This experimental study sought to determine whether LD filters inserted in the CPB circuit before cardioplegic arrest and in the BCP circuit during arrest w o u l d decrease ischemic myocardial damage. Methods. In 20 pigs, the second and third diagonal vessels were occluded for 90 minutes, followed by 45 minutes of BCP arrest and 180 minutes of reperfusion on CPB. In 5 pigs, LD filters were inserted in both the CPB and BCP circuits (LD-CPB+BCP). Five pigs had LD

during BCP (LD-BCP), 5 pigs had LD during CPB (LDCPB), and 5 pigs had no LD. Ischemic damage was assessed by wall motion scores using two-dimensional echocardiography and the area of necrosis/area of risk. Results. The LD-CPB and LD-CPB+BCP groups had the highest wall motion scores and the lowest area of necrosis/area of risk. The addition of LD to BCP alone did not significantly alter wall motion scores or the area of necrosis/area of risk. Conclusion. Leukocyte depletion filters significantly reduce ischemic damage during acute surgical revascularization and appear to be most effective w h e n placed in the CPB circuit before cardioplegic arrest.

nterest has focused recently on the role of leukocytes, particularly the neutrophil, as mediators of myocardial reperfusion injury [1, 2]. In experimental studies, leukocyte depletion (LD) of reperfusate blood has been shown to decrease myocardial necrosis after reperfusion of acutely ischemic m y o c a r d i u m [2, 3-7], after reperfusion of donor hearts for transplantation [8], and after p r o l o n g e d storage for heart [9] a n d lung [10] transplantation. However, Kofsky and co-workers [11] found that although LD of u n m o d i f i e d blood was beneficial after the reperfusion of acutely ischemic m y o c a r d i u m , it was inferior to the level of myocardial protection obtained w h e n blood cardioplegia (BCP) was given as the initial reperfusate. This suggested that LD m a y not confer any additional protection to hearts protected with BCP techniques. However, recent clinical studies by Chiba and associates [12] and Sawa and colleagues [13] showed that LD in patients u n d e r g o i n g urgent but not elective coronary artery bypass grafting (CABG) p r o v i d e d less myocardial damage. This implies that patients requiring CABG for acutely ischemic m y o c a r d i u m are the most likely to benefit from LD. This e x p e r i m e n t a l study was conducted to define further the role of LD during the revascularization of acutely

ischemic myocardium. Because studies in h u m a n s have shown that neutrophil activation begins i m m e d i a t e l y after the initiation of c a r d i o p u l m o n a r y bypass (CPB) [14], we sought to d e t e r m i n e w h e t h e r inserting a leukocytedepleting filter at the b e g i n n i n g of bypass, rather than after aortic unclamping, would diminish the degree of myocardial necrosis and stunning. Furthermore, we were interested in investigating the role of LD of BCP and d e t e r m i n i n g w h e t h e r leukocyte-depleted BCP given in an a n t e g r a d e - r e t r o g r a d e technique w o u l d confer any additional protection d u r i n g the revascularization of acutely ischemic myocardium. Finally, we w i s h e d to d e t e r m i n e w h e t h e r inserting leukocyte-depleting filters in both the CPB and BCP circuits would result in more complete protection than when each filter was used independently.

I

Accepted for publication Aug 4, 1995. Presented in part at the For~'-fourth Annual Scientific Session of the American College of Cardiology, New Orleans, LA, March 19 23, 1995. Address reprint requests to Dr Lazar, Department of Cardiothoracic Surgery, The Boston University Medical Center Hospital, 88 E Newton St, B404, Boston, MA 02118. © 1995 by The Society of Thoracic Surgeons

(Ann Thorac Surg 1995;60:1745-8)

Material and Methods

Preparation and Treatment Groups Twenty adult pigs (weight 28 to 35 kg) were p r e m e d i cated with intramuscular m o r p h i n e sulfate (2 mg/kg), anesthetized with a-chloralose (75 mg/kg), a n d placed on positive pressure ventilation. After m e d i a n sternotomy, catheters were placed in the aorta and left femoral vein for monitoring arterial pressure a n d a d m i n i s t e r i n g fluids. The azygos vein was ligated a n d a 9F retrograde cardioplegia catheter (DLP, Inc, G r a n d Rapids, MI) was inserted into the orifice of the coronary sinus via a pursestring in the right atrium. The animals were then a d m i n i s t e r e d h e p a r i n (3 mg/kg), and the second and 0003-4975/95/$9.50 SSD! 0003-4975(95)00737-7

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LAZAR ET AL LEUKOCYTE DEPLETION DURING CARDIOPLEGIA

third diagonal coronary arteries were occluded for 90 m i n u t e s with snares placed just distal to the takeoff from the left anterior coronary artery. Intravenous lidocaine was used to control ventricular arrhythmias, a n d ventricular fibrillation was treated with electrical defibrillation. After the 90-minute period of coronary occlusion, all animals were placed on CPB (Sarnes M e m b r a n e Oxygenator; Sarnes, Inc., A n n Arbor, MI), with a 17F c a n n u l a in the femoral artery a n d a 36F venous return catheter in the right atrium. A 24F catheter was inserted into the left atrium so that volume could be infused to vary the left ventricular end-diastolic pressure. Mean arterial blood pressure ranged from 65 to 70 m m Hg, and p u m p flow was kept at 80 mL • kg ~ • m i n ~. The hematocrit ranged from 25% to 28%, a n d pH was m a i n t a i n e d between 7.38 a n d 7.42. After the animals were placed on CPB, the hearts were arrested for 45 m i n u t e s with antegrade-retrograde BCP (temperature = 4°C, hematocrit = 16%, K = 30 mEq/L, pH = 7.6) administered every 20 m i n u t e s and supplem e n t e d with topical hypothermia. After the period of cardioplegic arrest, the aorta was unclamped, the coronary snares were released, and all hearts were reperfused by CPB at 37°C for 180 minutes. T r e a t m e n t Groups

After the institution of CPB, the animals were divided into four treatment groups. In the LD-CPB group (n = 5), a Pall LG6 arterial line LD filter (Pall Biomedical Inc, Glen Cove, NY) was inserted in the CPB circuit. In the LD-BCP group (n = 5), a Pall Blood Cardioplegia Leukocyte-Depleting BC IB Filter (Pall Biomedical Inc) was inserted in the BCP circuit. The LD-CPB+BCP group (n = 5) received a Pall LG6 arterial line LD filter inserted in the CPB circuit and a BC IB LD filter inserted in the BCP circuit. Finally, in the no-LD group (n - 5), no LD filters were inserted in either the CPB or BCP circuit. M e a s u r e m e n t s and Statistical A n a l y s e s

Electrocardiographic leads were placed to measure heart rate and to monitor electrical activity during cardioplegic arrest. A piezoelectric Mikro-Tip catheter pressure transducer (Millar Instruments, Houston, TX) was inserted into the apex of the left ventricle to measure left ventricular end-diastolic pressure. Systemic body temperature was m e a s u r e d using a rectal temperature probe (Yellow Springs Instruments, Yellow Springs, CO). Peripheral neutrophil counts were d e t e r m i n e d m a n u ally and expressed as the cell count × 103/mL. Neutrophils were m e a s u r e d before CPB and at 10, 60, a n d 180 minutes during CPB and averaged for each experimental group. Ecbocardiographic short-axis and long-axis sections were used to determine wall motion changes in the area of risk, as described previously [15]. A n u m e r i c score was used (4 = normal; 3 - mild hypokinesis; 2 = moderate hypokinesis; 1 = severe hypokinesis; 0 = akinesis; - 1 = dyskinesis) to determine the degree of wall motion abnormalities. These scores were averaged for the period of

A n n Thorac S u r g 1995;60:1745-8

4000 -

3000-" E 2000: 8 -----1000

*p<.05 from NOLD +p.O.05 from LD-BCP o NOLD D LO-BCP ~, LD-CPB • LD-CPB+BCP

5oo

Z

_+

_

PRECPB

10

*+.

~+

*+~ ~*+

60

180

CPB(min)

Fig 1. Granulocyte count. Lowest neutrophil counts with CPB are seen in the LD-CPB and LD-CPB+BCP groups. (BCP - blood cardioplegia; CPB = cardiopulmonary bypass; LD leukocyte depletion.)

coronary artery occlusion a n d reperfusion for all the experimental groups. The areas of risk a n d necrosis were d e t e r m i n e d by histochemical staining techniques, as described previously [15]. Statistical evaluations a m o n g the four experimental groups were performed using analyses of variance. Data were expressed as the m e a n -+ standard error and were considered significant at p value of less than 0.05. All animals received h u m a n e care in compliance with the " G u i d e for the Care a n d Use of Laboratory A n i m a l s " published by the National Institutes of Health (NIH publication 85-23, revised 1985).

Results The results are s u m m a r i z e d in Figures I to 3. Before CPB, the neutrophil counts were similar in all groups (see Fig 1). After 10 minutes of CPB, neutrophil counts fell significantly in all four groups. The lowest neutrophil counts were seen in the LD-CPB (89 ± 9 cells × 103/mL) a n d the LD-CPB+BCP (100 -+ 11 cells x 103/mL) groups. This trend c o n t i n u e d after 180 m i n u t e s of CPB. Hearts in the LD-CPB group had significantly lower neutrophil counts

~4-

020

%

1-

-1

A LD-CPB • LD-CPE~+BCP ~'p*.05 from NOLD +p<.05 from LD-BCP I

Preischemia

I

90min Coronary Occlusion

I

60min

I

180min

Reperfusion

Fig 2. Wall motion scores. The highest wall motion scores during reperfusion were seen in hearts that had LD filters added during CPB. (BCP blood cardioplegia; CPB - cardiopulmonary bypass; LD = leukocyte depletion.)

A n n Thorac S u r g 1995;60:1745-8

LAZAR ET AL LEUKOCYTE DEPLETION DURING CARDIOPLEGIA

100"

*p<.05 from NOLD

80'

+p*.05 from LD-BCP ~

60'

o

40' "I" ~+

<

~+

20-

NOLD

LD-BCP

"11

LD-CPB

LD-CPB+BCP

Fig 3. Areas of necrosis. The highest area of necrosis was seen in the No-LD and LD-BCP hearts. Hearts that had LD filters placed during CPB (LD-CPB; LD-CPB+BCP) had significantly smaller areas of necrosis. (BCP = blood cardioplegia; CPB cardiopulmonary bypass; LD = leukocyte depletion.)

(96 -+ 19 cells × 103/mL; p < 0.05) than those in the no-LD and LD-BCP groups. The addition of LD filters to both the BCP and CPB circuits did not result in a significant decrease in neutrophil counts (87 _+ 12 cells × 103/mL) compared with the LD-CPB group. These changes in neutrophil counts were reflected in the wall motion scores (see Fig 2) and the area of necrosis (see Fig 3). All groups had normal wall motion scores in the area of risk before ischemia and showed significant depression after 90 minutes of coronary occlusion (see Fig 2). Wall motion scores were significantly (p < 0.05) higher in the LD-CPB (3.6 + 0.1) and LD-CPB+BCP (3.4 + 0.2) hearts compared with the no-LD and LD-BCP groups after 180 minutes of reperfusion. The area of necrosis in the four groups is summarized in Figure 3. The highest area of necrosis was seen in the no-LD group (34% _+ 2%). Adding the LD filter to BCP resulted in a small decrease in the area of necrosis (29% _+ 1%). Hearts that received LD filters in the CPB circuit (LD-CPB) had a significantly (p < 0.05) lower area of necrosis than the no-LD and LD-BCP groups (17% _+ 1%). The addition of LD filters to both the cardioplegia and CPB circuits (LD-CPB+BCP) did not result in any significant improvement in the area of necrosis (19% ± 1%). Comment The renewed interest in LD has led to the development of commercial leukocyte filters that can accept blood flows required during extracorporeal support with relatively low pressure gradients. Currently available filters can remove at least 70% of neutrophils while preferentially sparing lymphocytes and platelets [16]. Earlier experimental studies without CPB demonstrated that the initiation of LD immediately upon reperfusion significantly decreased infarct size [2, 4-7]. However, Bryne and co-workers [5] and Kofsky and associates [11] noted that although the area of necrosis was decreased in neutrophil-depleted hearts, systolic dysfunction indicative of

1747

myocardial stunning persisted. There are several studies suggesting that LD may be more effective in preventing stunning if it is used as soon as CPB is instituted. Faymonville and co-workers [14] measured myeloperoxidase levels, a sensitive marker of neutrophil activation, in 15 patients undergoing elective CABG. Myeloperoxidase levels rose immediately upon the institution of bypass and increased further when systemic cooling was begun. Neutrophils that are activated just before hypothermic arrest can have a significant impact on reperfusion hemodynamics. Myers and colleagues [17] studied the effects of giving activated neutrophils to Langendorffperfused rabbit hearts before 40 minutes of global hypothermic arrest followed by normothermic reperfusion. Hearts receiving activated neutrophils showed a persistent and significant depression in ventricular force and the rate of ventricular tension development and relaxation, as well as a significant increase in coronary vascular resistance. Westlin and Mullane [21 showed that LD improved segmental shortening during reperfusion after 15 minutes of left anterior descending coronary artery occlusion in an experimental model. However, when blood was made leukocyte-free 30 minutes before ischemia, there was a further significant increase in systolic shortening, suggesting less "stunning." Earlier institution of LD during CPB could conceivably result in more circulating leukocytes later in the reperfusion period. This issue was studied by Wilson and co-workers [3], who looked at the rate of leukocyte reaccumulation after LD early during CPB. Neonatal pigs underwent 90 minutes of hypothermic cardioplegic arrest, followed by 6 hours of reperfusion. Neutrophil counts fell to 0.8% of control during early reperfusion but by 6 hours climbed to 68%. Despite this, leukocyte-depleted hearts had significantly greater preservation of left ventricular performance, systolic function, and compliance, and decreased water content and myeloperoxidase activity. Hence, although early removal of leukocytes resulted in a return toward control levels of neutrophils during reperfusion, there was still significant improvement in myocardial performance and less stunning. Our own results support the concept of early LD during the revascularization of acutely ischemic myocardium. Hearts receiving LD filters immediately upon instituting CPB had significantly less myocardial stunning as represented by higher wall motion scores (see Fig 2) and less necrosis in the area at risk (see Fig 3). Our study supports the earlier work of Kofsky and co-workers [11] by showing that the addition of leukocyte-depleting filters to BCP solutions failed to improve wall motion scores or decrease the area of necrosis. Furthermore, the addition of leukocyte-depleting filters to both the BCP and CPB circuits failed to result in any better protection than that achieved by LD of the CPB circuit alone. Our results also imply that the removal of activated leukocytes upon the institution of CPB in acutely ischemic myocardium confers significant protection from myocardial stunning and extension of infarct size. Several clinical studies suggest that LD may play a role during clinical cardiac operations [12, 13]. Chiba and

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LAZARET AL LEUKOCYTEDEPLETIONDURINGCARDIOPLEGIA

associates [12] used leukocyte-depleting filters in the bypass circuits of 26 patients u n d e r g o i n g CABG. The leukocyte-depleted patients had significantly decreased enzyme leakage, less catecholamine use, and higher cardiac indices in the early postoperative period [12]. Sawa a n d co-workers [13] further defined the role of leukocyte-depleting filters in a study involving both elective and emergent CABG patients. Leukocyte-depleting filters were a d d e d to terminal BCP solutions. Leukocyte depletion did not confer any added protection to hearts u n d e r g o i n g elective CABG. However, in the emergent group, LD significantly lowered peak CK-MB levels a n d decreased the need for inotropic support. Our own previous studies have shown that d u r i n g the revascularization of acutely ischemic myocardium, such as after a failed percutaneous t r a n s l u m i n a l coronary angioplasty, ischemic damage may occur before the institution of CPB a n d persist u p o n reperfusion [18]. We hypothesize based on the results of this study that LD deployed at the initiation of CPB may have the greatest benefit on the revascularization of acutely ischemic myocardium. In addition to decreasing ischemic myocardial damage, leukocyte-depleting filters may decrease the p u l m o n a r y dysfunction associated with CPB, resulting in better arterial oxygenation, decreased p u l m o n a r y vascular resistance, a n d decreased lung water accumulation [19]. Clinical studies are warranted to attempt to determ i n e whether the favorable effects of LD in these experimental studies will translate into lower morbidity and mortality rates and shorter hospital stay for unstable and e m e r g e n t CABG patients. Supported in part by a grant from the Heart Disease Research Foundation. The secretarial support of Mrs Ellie LaBombard in the preparation of this manuscript is gratefully acknowledged.

References 1. Weiss SJ. Tissue destruction by neutrophils. N Engl J Med 1989;320:365-76. 2. Westlin W, Mullane KM. Alleviation of myocardial stunning by leukocyte and platelet depletion. Circulation 1989;80: 1828-36. 3. Wilson IC, Gardner TJ, DiNatale JM, Gillinov AM, Curtis WE, Cameron DE. Temporary leukocyte depletion reduces ventricular dvsfunction during prolonged postischemic reperfusion. J ~Fhorac Cardiovasc Surg 1993;106:805-10. 4. Litt MR, Jeremy RW, Weisman HF, Winkelstein JA, Becker LC. Neutrophil depletion limited to reperfusion reduces myocardial infarct size after 90 minutes of ischemia. Circulation 1989;80:1816-27.

Ann Thorac Surg 1995;60:1745-8

5. Byrne JG, Appleyard RF, Lee CC, et al. Controlled reperfusion of the regionally ischemic myocardium with leukocytedepleted blood reduces stunning, the no-reflow phenomenon, and infarct size. J Thorac Cardiovasc Surg 1992;103: 66-72. 6. Sawa Y, Nakano S, Shimazaki Y, Nishimura M, Kuratani T, Matsuda H. Myocardial protective effect and its mechanism of leukocyte-depleted reperfusion in neonatal rabbit hearts. Ann Thorac Surg 1994;58:1386-91. 7. Kawata H, Sawatari I, Mayer E. Evidence for the role of neutrophils in reperfusion injury after cold cardioplegic ischemia in neonatal lambs. J Thorac Cardiovasc Surg 1992; 103:908-18. 8. Byrne JG, Smith WJ, Murphy MP, Couper GS, Appleyard RF, Cohn LH. Complete prevention of myocardial stunning, contracture, low-reflow, and edema after heart transplantation by blocking neutrophil adhesion molecules during reperfusion. J Thorac Cardiovasc Surg 1993;104:1589-96. 9. Breda MA, Drinkwater DC, Laks H, et al. Prevention of reperfusion injury in the neonatal heart with leukocytedepleted blood. ] Thorac Cardiovasc Surg 1989;96:654-65. 10. Schueler S, DeValeria PA, Hatanaka M, et al. Successful twenty-four hour lung preservation with donor core cooling and leukocyte depletion in an orthotopic double lung transplantation model. J Thorac Cardiovasc Surg 1993;204:73-82. 11. Kofsky ER, Julia PL, Buckberg GD, Quillen JE, Acar C. Studies of controlled reperfusion after ischemia XXII. Reperfusate composition: effects of leukocyte depletion of blood and blood cardioplegic reperfusates after acute coronary occlusion. J Thorac Cardiovasc Surg 1991;350-9. 12. Chiba Y, Muraoka R, Ihaya A, Morioka K, Sasaki M, Vesaka T. Leukocyte depletion and prevention of reperfusion injury during cardiopulmonary bypass: a clinical study. Cardiovasc Surg 1993;1:350-6. 13. Sawa Y, Matsuda H, Shimazaki Y, et al. Evaluation of leukocyte-depleted terminal blood cardioplegic solution in patients undergoing elective and emergency coronary artery bypass grafting. J Thorac Cardiovasc Surg 1994;108:1125-31. 14. Faymonville ME, Pincemail J, Duchateau J, et al. Myeloperoxidase and elastase as markers of leukocyte activation during cardiopulmonary bypass in humans. J Thorac Cardiovasc Surg 1991;102:309-17. 15. Lazar HL, Yang XM, Rivers S, Treanor P, Shemin RJ. Role of percutaneous bypass in reducing infarct size after revascularization for acute coronary insufficiency. Circulation 1991; 84(Suppl 3):416-21. 16. Gourlay T, Fleming J, Taylor KM. Laboratory evaluation of the Pall LG6 leukocyte depleting arterial line filter. Perfusion 1992;7:131-40. 17. Myers ML, Webb C, Moffat M, McIver D, DelMaestro RD. Activated neutrophils impair rabbit heart recovery after hypothermic global ischemia. Ann Thorac Surg 1992;53: 247-52. 18. Lazar HL, Faxon DP, Paone G, et al. Changing profiles of failed coronary angioplasty patients: impact on surgical results. Ann Thorac Surg 1992;53:269-73. 19. Bando K, Pillai R, Cameron DE, et al. Leukocyte depletion ameliorates free radical-mediated lung injury after cardiopulmonary bypass. J Thorac Cardiovasc Surg 1990;99:873-7.