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Eur J Cardiothorac Surg 2000;18:90-97
© 2000 Elsevier Science NL

Leukocyte-depleted reperfusion after long cardioplegic arrest attenuates ischemia–reperfusion injury of the coronary endothelium and myocardium in rabbit hearts

Department of Thoracic and Cardiovascular Surgery, Saga Medical School, 5-1-1 Nabeshima, Saga City, Saga 849-8501, Japan

Received 6 September 1999; received in revised form 9 March 2000; accepted 13 March 2000.

Corresponding author. Tel.: +81-952-34-2345; fax: +81-952-34-2061
e-mail: okazaki{at}post.saga-med.ac.jp

	Abstract

Top Abstract 1. Introduction 2. Methods 3. Results 4. Discussion References

 
Objective: Cardiopulmonary bypass activates leukocytes, which should injure the coronary endothelium and myocardium during reperfusion especially after long cardioplegic arrest with long cardiopulmonary bypass time. The present study was designed to determine the protective efficacy of leukocyte-depleted reperfusion in blood-perfused parabiotic isolated rabbit hearts as a surgically relevant model with long cardioplegic arrest. Methods: Each isolated rabbit heart, with a latex balloon inserted in the left ventricle, was parabiotically blood-perfused using a modified Langendorff column. The left ventricular developed pressure (DP), rate of pressure development (dP/dT), and coronary flow with a left ventricular end-diastolic pressure of 10 mmHg were measured before ischemia and after 15, 30, 45, and 60 min reperfusion after 4 h cardioplegic arrest kept at 20°C (control, n=10). Leukocyte-depleted reperfusion was done in the test group (n=10). The endothelium of the coronary artery was observed by scanning electron microscopy (SEM) with percent injured area of endothelial cells measured to evaluate the extent of endothelial ischemia–reperfusion injury. Results: The control hearts showed 53.3, 54.3, 48.4, and 39.0% recovery of DP compared to the pre-ischemia baseline data at 15, 30, 45, and 60 min after reperfusion began respectively. Leukocyte-depleted reperfusion enhanced the recovery of DP at 45 min (81.3%, P=0.0021) and 60 min (85.8%, P=0.0005) after reperfusion compared with that in the control group. The control hearts revealed 58.8%, 59.8%, 52.6%, and 43.4% recovery of dP/dT compared to the pre-ischemia baseline data at 15, 30, 45, and 60 min after reperfusion began, respectively. Leukocyte-depleted reperfusion also enhanced the recovery of dP/dT at 45 min (93.2%, P=0.0071) and 60 min (98.8%, P=0.0011) after reperfusion compared with that in the control group. There was also improvement of the recovery of coronary flow by leukocyte-depleted reperfusion (97.2%) compared with that in the control group (58.3%) after 60 min reperfusion (P=0.0121). Scanning electron microscopy showed that 69.7% of coronary endothelial cells were morphologically injured in the control group. In contrast, leukocyte-depleted reperfusion prevented the extent of coronary endothelial damage with less injured area (0.5%, P=0.0002). Conclusions: Leukocyte-depleted reperfusion improved functional recovery with reduced coronary endothelial injury after long cardioplegic arrest.

Key Words: Leukocyte-depleted reperfusion • Ischemia–reperfusion injury • Leukocyte • Endothelium • Myocardium

	1. Introduction

Top Abstract 1. Introduction 2. Methods 3. Results 4. Discussion References

 
Although great advances in cardiac protection during cardiac surgery have been achieved, cardiac surgeons still encounter post-cardiotomy cardiogenic shock especially in cases with prolonged cardioplegic arrest. Cardiopulmonary bypass (CPB) induces inflammatory changes with leukocytes activated [1–6], which injure the myocardium and coronary endothelium during reperfusion. It is therefore quite important to reduce myocardial, as well as endothelial, ischemia–reperfusion injury associated with activated leukocytes, especially in cases of long cardioplegic arrest with long CPB.

Recently, leukocyte-depleted reperfusion has been reported as an effective method to reduce ischemia–reperfusion injury in neonatal hearts initially arrested with cold cardioplegic solution, then stored [7,8], or regionally ischemic myocardium [9,10]. The efficacy of leukocyte-depleted reperfusion were also clinically determined in heart transplantation [11,12] or emergency coronary artery bypass grafting [13].

In the present study, the protective effect of the leukocyte-depleted reperfusion on the myocardium and coronary endothelium after a 4-h cardioplegic arrest was determined using a parabiotic isolated blood-reperfused Langendorff system. The coronary endothelial injury was also analyzed morphologically with scanning electron microscopy (SEM) to learn time course of the endothelial reperfusion injury, as well as to compare reperfusion injury morphologically between the groups.

	2. Methods

Top Abstract 1. Introduction 2. Methods 3. Results 4. Discussion References

 
2.1. Animal care
All of the animals involved in this study received humane care in compliance with the ‘Guide for the Care and Use of Laboratory Animals’ published by the National Institutes of Health (NIH Publication No.86-23, revised 1985) and with the European Convention on Animal Care. All procedures were approved by the Animal Research Committee of the Saga Medical School.

2.2. Preparation of the support animals
Adult male Japanese white rabbits weighing 3.0–3.4 kg were used as support animals. The support animal was anesthetized intramuscularly with ketamine (50 mg/kg) to establish a vascular access line into the left ear vein for giving pharmacologic agents and lactated Ringer's solution. The animal was then deeply anesthetized with pentobarbital sodium (5–7 mg/kg, i.v.) and pancuronium bromide (0.08 mg/kg, i.v.) to make a tracheostomy for mechanical ventilation support (model SN-480-5, Shinano. Co., Tokyo, Japan) with 100% oxygen throughout the experiment. After heparinization (500 units/kg) the right femoral artery was cannulated for arterial blood pressure monitoring using a pressure transducer (model RM-6000, Nihon Kohden, Tokyo, Japan). The right carotid artery and the left jugular vein were cannulated to establish extracorporeal circulation. Anesthesia was maintained by approximately hourly additional intramuscular administration of ketamine (25 mg/kg). Arterial blood gas and hemoglobin concentrations of the support animals were analyzed (ABL510, Radiometer Medical, Copenhagen, Denmark) every 30 min throughout the experiments.

2.3. Preparation of the donor animals
Adult male Japanese white rabbits weighing 2.7–3.0 kg were used as heart and blood donors. The donor animal was anesthetized and ventilated in the same way as the support rabbit with heparinization (500 U/kg) achieved. To save the number of animals used, 45 ml of blood, which was used to prime the modified Langendorff column, was retrieved at first with the same volume of lactated Ringer's solution infused to prevent hypovolemic shock of the donor animal. Cardiectomy was then performed through a median sternotomy.

2.4. Establishment of the blood-perfused, parabiotic, isolated heart Langendorff system
A modified Langendorff column (Fig. 1) , which was composed of glass thermoregulators and polyvinyl chloride tubes, was primed with blood from the heart donor rabbit and heparinized (1500 U/column) lactated Ringer's solution. The carotid artery cannula was connected to the pump (model MP-3, Rikakikai Co. Ltd., Tokyo, Japan). Arterial blood was actively drained using this pump and was fed into a modified Langendorff apparatus. The height of the column was set at 80 cmH₂O. The venous flow from the Langendorff column was returned to the jugular vein of the support animal using the same type of pump. The systolic arterial pressure of the support animal was maintained above 70 mmHg and the central venous pressure was maintained between 0 and 2 cmH₂O. The perfusion temperature was maintained at 37°C.

View larger version (29K): [in this window] [in a new window]   Fig. 1. Schematic illustration of the modified Langendorff column used. Arterial blood from the support animal was used to perfuse the isolated heart. The height of the column was set at 80 cmH2O. A leukocyte removal filter was used to reperfuse the isolated heart with leukocyte-depleted blood.

2.5. Experimental protocol
After a 30-min equilibration period, saline solution was infused into the intraventricular latex balloon to generate end-diastolic pressure (EDP) of 10 mmHg. Coronary flow rate, the left ventricular developed pressure (DP), and the rate of positive pressure development (dP/dT) at an EDP of 10 mmHg were measured as baseline data. After acquisition of the baseline data the fluid in the latex balloon was adjusted to obtain an EDP of 0 mmHg.

To achieve cardioplegic arrest similar to that of a clinical procedure, 50 ml of St. Thomas’ Hospital solution (in mM: NaCl 110.0, NaHCO₃ 10.0, KCl 16.0, MgCl₂ 16.0, CaCl₂ 1.2) at 4°C was infused from a height of 80 cmH₂O through a separate column. The effluent from the coronary sinus was collected and discarded from the circuit. After cardiac arrest the hearts were immersed in saline solution at 20°C for 4 h. The heart was given additional cardioplegic solution (25 ml) every 30 min during the 4 h of global ischemia. Blood was continuously recirculated between the support rabbit and the circuit of Langendorff apparatus at a flow rate of 10 ml/min to allow the potential activation of leukocytes as in a clinical CPB operation.

After 4 h of cardioplegic arrest, heparin (1500 units/column) was added. Then, the heart was reperfused with whole blood for 60 min in the control group (n=10) or reperfused with leukocyte-depleted blood through a leukocyte removal filter (Sepacell; Asahi Medical, Tokyo, Japan) for 60 min in the test group (n=10). Effective leukocyte depletion through the filter was confirmed by sampling blood for leukocyte counting (Celltac MEK-5257, Nihon Kohden). The left ventricular pressure–volume relationships and coronary flow rate were measured 15, 30, 45, and 60 min after reperfusion began. Blood gas analysis was repeated every 30 min to ensure the stability of the support animals throughout the experiments. At the conclusion of the experiment the cardioplegic solution was perfused into the isolated heart. Then the tissue was fixed by perfusing 2.5% glutaraldehyde in 0.1 M cacodylate buffer with 3% sucrose at a perfusion pressure of 80 cmH₂O.

2.6. Observation of the coronary endothelium by scanning electron microscopy
The heart was stored in the same fixative until it was studied. After rinsing with 0.1 M cacodylate buffer, the heart was dehydrated through an ethanol series and freeze-dried. The tissue specimen including the endothelial surface of the epicardial coronary artery was coated with gold (IB-3 ion coater, Eiko Ltd., Mito, Japan), and then observed by SEM (JSM-5200LV, JEOL Ltd., Tokyo, Japan).

The support animal heart was also retrieved, then prepared for SEM study in a same fashion. The blood-contacting surface of the modified Langendorff circuit was also prepared for SEM observation to detect the deposited blood cells potentially activated by extracorporeal circulation.

To observe the time course of endothelial damage caused by leukocytes in the control settings, additional experiments were done to retrieve the hearts after 4 h of cardioplegic arrest without reperfusion (n=5), as well as 15 (n=5), 30 (n=4), and 45 min (n=4) whole blood reperfusion after 4 h of cardioplegic arrest. The same preparation for SEM study was done on each retrieved heart.

To achieve quantitative analysis of the coronary endothelial damage, the percentage of damaged area was calculated using a computer image analysis system (MacSCOPE, Mitani Co. Ltd., Fukui, Japan) in each SEM specimen.

2.7. Statistical analysis
Results are expressed as the mean±standard deviation (SD). Statistical analyses were performed by the Mann–Whitney test to compare the data between the groups. Differences were considered significant at the level of P<0.05. In analyses of percent recoveries of DP, dP/dT and coronary flow, multiple comparisons were done using single Mann–Whitney tests with Bonferroni correction as post hoc comparisons if applicable.

	3. Results

Top Abstract 1. Introduction 2. Methods 3. Results 4. Discussion References

 
3.1. Condition of the support animals, baseline data, and effective leukocyte removal
The conditions of the support animals were stable throughout the experiments. There were no significant differences between the two groups in the baseline systolic blood pressures, blood gas data, or hemoglobin concentrations of the support animals (Table 1). During reperfusion, no significant differences were found in these values between the groups (Table 2).

View larger version (39K): [in this window] [in a new window]   Fig. 2. Percentage recoveries of developed pressure during reperfusion after 4 h of cardioplegic arrest. Leukocyte-depleted reperfusion significantly improved the percent recovery of developed pressure at 45 (P=0.0021) and 60 min (P=0.0005) after reperfusion began. P-values were calculated by the Mann–Whitney test. Control, whole blood reperfusion; leukocyte depleted, leukocyte-depleted blood reperfusion.

View larger version (39K): [in this window] [in a new window]   Fig. 3. Percentage recovery of dP/dT during reperfusion after 4 h of cardioplegic arrest. Leukocyte-depleted reperfusion significantly improved the percent recovery of dP/dT at 45 (P=0.0071) and 60 min (P=0.0011) after reperfusion began. P-values were calculated by the Mann–Whitney test. Abbreviations are the same as for Fig. 2.

View larger version (43K): [in this window] [in a new window]   Fig. 4. Percentage recovery of coronary flow during reperfusion after 4 h of cardioplegic arrest. Leukocyte-depleted reperfusion significantly improved the percent recovery of dP/dT at 60 min (P=0.0121) after reperfusion began. P-values were calculated by the Mann–Whitney test. Abbreviations are the same as for Fig. 2.

View larger version (93K): [in this window] [in a new window]   Fig. 5. Scanning electron micrographs of the coronary endothelium. (A) The isolated heart after reperfusion with whole blood for 60 min after 4 h of cardioplegic arrest. Many endothelial cells were delaminated. Blood cells were deposited mainly on the delaminated area (x1500). (B) The isolated heart with leukocyte-depleted reperfusion without endothelial delaminations (x2000). (C) In the support animal hearts, endothelium appeared almost normal in spite of the same whole blood perfusion (x1500).

View larger version (133K): [in this window] [in a new window]   Fig. 6. Scanning electron micrographs of the coronary endothelium. (A) The isolated heart just after 4 h cardioplegic arrest without reperfusion (x1500). (B) The isolated heart after 15 min of whole blood reperfusion. The endothelial damage was already detected (x1500).

	4. Discussion

Top Abstract 1. Introduction 2. Methods 3. Results 4. Discussion References

 
Leukocytes are known to contribute to both myocardial and endothelial damage after reperfusion of an ischemic heart [14–16]. In the present blood-perfused parabiotic isolated rabbit hearts with 4-h cardioplegic arrest, it is noteworthy that, despite the fact that cardioplegic arrest was achieved by infusion every 30 min of cold St. Thomas’ Hospital solution, the severe coronary endothelial injury was morphologically detected by SEM after whole blood reperfusion with myocardial contractility impaired with less than 50% recovery of left ventricular developed pressure. SEM analysis of the coronary endothelium in the present study also demonstrated that the morphologically detectable damage was not seen in the hearts after 4 h of cardioplegic arrest without reperfusion or in the support rabbit hearts with the same whole blood continuously perfused without ischemia. Tsao et al. [14] reported only functional endothelial damage, which occurred within 2.5–5 min of reperfusion, after occlusion of the left anterior descending artery for 90 min and reperfusion for up to 270 min without morphological damage to the surface of the endothelium observed by SEM. In the present study, extracorporeal circulation during cardioplegic arrest potentially activated the leukocytes, which might have enhanced both the coronary endothelium and myocardium injury during reperfusion.

Although we did not quantify the degree of inflammatory reaction of this experiment by measuring cytokines or various inflammatory parameters, it could be considered that this extracorporeal circulation of the modified Langendorff circuit for more than 4 h activated the contact system with platelets and a few leukocytes deposited on the blood-contacting surfaces of the circuit, which were observed by SEM. We tried to establish a clinically relevant model simulating a case requiring long cardioplegic arrest. We still, however, note some limitations of this experiment in which actual CPB was not used.

In the present study, the protective efficacy of leukocyte-depleted reperfusion on the coronary endothelium and myocardial contractility after long cardioplegic arrest was obviously shown in a surgically relevant model in which cardioplegic arrest was achieved in a conventional fashion without intentional myocardial ischemia. Not only leukocytes themselves but also chemical mediators associated with inflammation in plasma play an important part in ischemia–reperfusion injury [17,18]. In the present study, only leukocyte-depleted reperfusion improved the myocardial contractility over time in spite of reperfusion of plasma after inflammatory responses associated with the more than 4 h of extracorporeal circulation. This evidence supports the evidence that leukocytes are a major factor in reperfusion injury. Post-cardiotomy cardiogenic shock associated with prolonged cardioplegic arrest may be prevented by appropriate leukocyte-depleted reperfusion.

How long should the leukocyte-depleted reperfusion be continued? In the present study, the leukocyte-depleted reperfusion was continued for 60 min after reperfusion began. Then, the coronary endothelium and myocardium were protected from reperfusion injury associated with leukocytes. In the clinical setting, only temporary leukocyte depletion can be done practically. Byne and colleagues [10] demonstrated the coronary endothelial and myocardial protective efficacy of 20-min leukocyte-depleted reperfusion followed by an additional 3 h of whole blood reperfusion in the regionally ischemic myocardium. Only 15 min of whole blood reperfusion caused morphologically detected reperfusion injury of the coronary endothelium after 4 h of cardioplegic arrest in the present study. There may be a possibility to prevent coronary endothelial reperfusion injury by an initial 15–20-min leukocyte-depleted reperfusion. Weyrich and colleagues [19] investigated the time course of coronary vascular endothelial adhesion molecule expression during reperfusion in the ischemic feline myocardium. They found that P-selectin was maximally expressed 20 min after reperfusion, while intercellular adhesion molecule-1 (ICAM-1) was significantly increased following 150 and 270 min of reperfusion. To consider the expression of ICAM-1 on the endothelium during reperfusion, up to 270 min of leukocyte-depleted reperfusion may be recommended. In any case, it is quite important to establish a practical method of leukocyte-depleted reperfusion after prolonged cardioplegic arrest, by which post-cardiotomy cardiogenic shock associated with leukocyte-mediated reperfusion injury can be prevented.

Heparin-bonded CPB surfaces decreased inflammatory reaction with reducing complement activation [20]. A broad-spectrum protease inhibitor, nafamostat mesilate, also modulated platelet, neutrophil and contact activation in simulated extracorporeal circulation [21]. These methods to decrease leukocyte activation caused by CPB could also improve coronary endothelial and myocardial reperfusion injury. A monoclonal antibody to ICAM-1 [22], as well as a monoclonal antibody to P-selectin [23], successfully attenuated coronary endothelial and myocardial reperfusion injury. Inhibition of cell adhesion between leukocytes and coronary endothelium may be a substitute for leukocyte-depleted reperfusion. To combine these methods, a better protective effect in ischemia–reperfusion injury may be achieved in the clinical setting.

In conclusion, whole blood reperfusion after 4 h of cardioplegic arrest caused morphologically detectable endothelial injury with myocardial contractility decreased. In contrast, leukocyte-depleted reperfusion significantly attenuated the coronary endothelial and myocardial reperfusion injury after 4 h of cardioplegic arrest in blood-perfused parabiotic isolated rabbit hearts, used as a surgically relevant model.

	Acknowledgments

 
The authors wish to express their profound gratitude to Dr Katsuhisa Horimoto, Laboratory of Mathematics, for his guidance on the statistical analysis; Mr Toshimi Tabata and Mr Shin-ichi Nakahara for their technical assistance; and Mr Kent E. Wika for his assistance in manuscript preparation.

	Footnotes

 
Presented at the 13th Annual Meeting of the European Association for Cardio-thoracic Surgery, Glasgow, Scotland, UK, September 5–8, 1999.

	References

Top Abstract 1. Introduction 2. Methods 3. Results 4. Discussion References

 

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This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Add to Personal Folders Download to citation manager Author home page(s): Yukio Okazaki Masafumi Natsuaki Tsuyoshi Itoh Permission Requests Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Okazaki, Y. Articles by Itoh, T. Search for Related Content PubMed PubMed Citation Articles by Okazaki, Y. Articles by Itoh, T.