Ann Thorac Surg 2000;70:1531-1535
© 2000 The Society of Thoracic Surgeons
Original articles: cardiovascular
Correlation between ICAM-1 and functional recovery of piglet myocardium with leukocyte-depleted reperfusion
Jeong Ryul Lee, MDa,
Jae Jin Han, MDb,
Jeong Wook Seo, MDa
a Department of Thoracic and Cardiovascular Surgery, and Pathology, Seoul National University Hospital, Seoul National University College of Medicine, Seoul National University Medical Research Center, Heart Research Institute, Seoul, South Korea
b Department of Thoracic and Cardiovascular Surgery, Ewha University Mokdong Hospital, Ewha University College of Medicine, Seoul, South Korea
Address reprint requests to Dr Lee, Department of Thoracic and Cardiovascular Surgery, Seoul National University College of Medicine, 28 Yongon-dong Chongro-gu, Seoul 110-744, Korea
e-mail: jrl{at}plaza.snu.ac.kr
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Abstract
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Background. Reperfusion injury involves leukocyte–endothelial interaction mediated by cell adhesion molecules. This study was designed to determine the time course of intercellular adhesion molecule-1 (ICAM-1) expression and the functional recovery of myocardium when reperfused with leukocyte depleted whole blood.
Methods. Sixteen neonatal piglet hearts were harvested and stored with 4°C cold University of Wisconsin Solution (UWS) for 12 hours. An ex vivo model consisting of an isolated working heart perfusion circuit, roller pumps, and a membrane oxygenator, was used for reperfusion. Atrial tissues were taken for staining ICAM-1. The stroke work index (SWI) was calculated during 4 hours of reperfusion. Two groups (group 1: reperfused with whole blood, group 2: with leukocyte depleted blood) were compared.
Results. The differences of ICAM-1 expression between group 1 and 2 were significant at 3 and 4 hours of reperfusion (p < 0.05). The differences of the mean stroke work indices were significant at 2, 3, and 4 hours after reperfusion (p < 0.05).
Conclusions. Leukocyte-depleted reperfusion attenuates the expression of ICAM-1 and reduces the time-dependent functional deterioration of the myocardium. These results suggest that adhesion molecule like ICAM-1 plays a major role in deteriorating myocardial function during the reperfusion, possibly by leukocyte-mediated inflammatory process.
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Introduction
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Leukocyte adhesion is an important step leading to ischemia-reperfusion injury of the myocardium. The donor heart myocardium and its function after transplantation could be also affected by adhesion molecule–induced reperfusion injury. Numerous studies both in vitro and in vivo have shown that neutrophils are activated by various cytokines and interact with the endothelium by rolling, firm adhesion, and transmigration to the tissue. Reports have shown that endothelial dysfunction characterized by a decreased production of an endothelium-derived relaxing factor occurs within 10 minutes after the onset of reperfusion. Endothelial dysfunction can also be caused by neutrophil adhesion to the coronary endothelial surface within 20 minutes after reperfusion as well as by neutrophil accumulation within the myocardium, which may occur 3 hours after reperfusion [1, 2]. Bevilacqua and colleagues indicated that ICAM-1 and E-selectin are upregulated by cytokines over a period of 2 to 4 hours by a process requiring protein synthesis [3]. In addition, Pearl and associates showed that leukocyte-depleted reperfusion of the transplanted human heart decreases biochemical evidence of reperfusion injury despite an ischemic time of less than 3 hours [4].
In our study, using an isolated working heart perfusion model consisting of a membrane oxygenator and roller pumps, we endeavored to determine the effect of reperfusion with leukocyte-depleted blood in 12-hour cold ischemic–stored neonatal piglet hearts on the expression of ICAM-1 in the myocardium. Stroke work index was used as a functional variable to evaluate the correlation between the ICAM-1 expression and the functional recovery of the myocardium.
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Material and methods
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Experimental group
The leukocyte-depleted reperfusion group (group 2, n = 8) included hearts that were reperfused with leukocyte-depleted whole blood after 12-hour cold ischemic storage. The control group (group 1, n = 8) included hearts that were reperfused with unmodified whole blood.
Harvest and storage
Neonatal piglets (aged 1 to 3 days) were anesthetized with intramuscular ketamine (100 mg/kg) and intubated with a 3.5F endotracheal tube through a tracheostomy. Mechanical ventilation (Servo Ventilator 900C Siemens-Elema) at an inspired oxygen fraction of 1.0, a tidal volume of 60 ml, and a rate of 20 breaths per minute. The chest was opened through a median sternotomy and the pericardium was opened to expose the heart. Ligation of the hemiazygos vein was performed. The descending aorta, left and right innominate arteries, and superior and inferior vena cavae were subsequently encircled, in that order. Heparin (300 U/kg) and cefazolin (30 mg/kg) were injected into the pulmonary artery. After distal ligation, the right innominate artery was cannulated with a 12-gauge angiocatheter and the pressure monitored. After ligation of the superior and inferior vena cavae and the venting of both ventricles, the descending aorta was cross-clamped and the heart arrested with the University of Wisconsin Solution (UWS), as shown in Table 1. A quantity of 100 ml of UWS was infused by means of an innominate artery cannula at a mean pressure of 60 mm Hg with simultaneous topical cooling. Cardiectomy was performed and the heart was immersed in 4°C cold UWS. The container was sealed, packed in ice, and stored for 12 hours.
Reperfusion
Reperfusion was achieved with an isolated, blood-perfused working heart model (Fig 1), which consisted of two roller pumps and a membrane oxygenator (Microsafe, Polystan Inc, Copenhagen, Denmark), a heated reservoir/bubble trap (Conducer Cardioplegia Heat Exchange, 3 mol/L, Sarns, Ann Arbor, MI), an afterload controlling tube column, and a preload controlling bowel. Blood for reperfusion was collected from an anesthetized adult pig (Yorkshire, body weight, > 50 kg) after injection of heparin(300 U/kg). For group 2, blood was filtered with a leukocyte filter (Sepacell R, Asahi Medical, Tokyo, Japan). The circuit was primed with 400 mL of whole blood and 100 mL of Hartman’s solution, to which glucose (2 g) and insulin (4 U) were added. The hematocrit was maintained at 25%. Reperfusion was started with a mean perfusion pressure of 60 mm Hg by means of an innominate cannula. After 20 minutes of nonworking perfusion, the circuit was converted into working mode. The descending aorta was connected to a column, the height of which was adjusted to a mean pressure of 60 mm Hg. The left atrial preload was set to 6 mm Hg by adjusting the level of the bowel.
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Fig 1. Diagram of the isolated working heart perfusion circuit. Donor blood, either passing through a leukocyte filter (group 2) or not passing through (group 1), is pumped to either the aortic root or a reservoir that supplies the left atrium. The afterload is set to 60 mm Hg by the height of the aortic column. Cardiac output is measured by timed collections of the coronary sinus effluent and overflow from the aortic column. (LF = leukocyte filter; CO = cardiac output; CSE = coronary sinus effluent; HR = heated reservoir; Ao = aorta; PA = pulmonary artery; RA = right atrium; LA = left atrium; RV = right ventricle; LV = left ventricle.)
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Biopsy and immunohistochemistry
Right atrial tissues for the adhesion molecule assay was taken at 0, 0.5, 1, 2, 3, and 4 hours after reperfusion. Biopsy specimens were snap-frozen and cryosections (4 µm thick) were washed in phosphate-buffered saline (PBS) and prediluted swine serum (Dako, Glostrup, Denmark). The sections were then incubated with the primary antibody (for ICAM-1: monoclonal antibody antihuman ICAM-1(CD54) (R&D, Gaithersburg, MD) diluted 1:100 in blocking serum) for 2 hours and washed in PBS. The specimens were then incubated with fluorescent isothiocyanate (FITC)-labeled secondary antibodies (for ICAM-1: FITC conjugated antimouse IgG [Dako] diluted 1:50 in blocking serum) for 30 minutes. After subsequent washes in phosphate-buffered saline with 0.05% Tween 20, the slides were counterstained with propodium iodide/antifade (Oncor, Gaithersburg, MD), and mounted in fluorescence mounting media (Dako). The sections were examined and scored on a scale of 0 to 4 (0 = no evidence of expression, 1 = trace, 2 = mild, 3 = moderate, and 4 = strong) by two of the authors and the results averaged in a semiquantitative manner (Fig 2). Photomicrographs were taken with a fluorescence microscope(AHBT3, VANOX, Olympus, Tokyo, Japan).
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Fig 2. Immunofluorescent micrograph of the myocardium using antibody against ICAM-1. A, Trace (+) immunoreaction (open arrowheads) of the venules and arterioles shows incomplete circles at the vascular endothelium. B, Moderate (+++) immunoreaction shows fluorescence (arrowheads) at the vascular endothelium.
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Myocardial water content
The wet weight of the excised myocardium (left ventricle) was measured and the myocardium was air-dried until a constant weight was achieved. Water content was expressed as percent wet/dry ratio.
where Wetwt = wet weight and DryWt = dry weight.
Evaluation of functional recovery
The function of left ventricle was assessed using a measurement of cardiac output (aortic column overflow) at a constant aortic pressure of 60 mm Hg and a mean atrial pressure of 6 mm Hg. The stroke work index was calculated as follows:
where SWI = stroke work index, MAP = mean aortic pressure, LAP = mean left atrial pressure, CO = cardiac output, HR = heart rate, and H Wt = heart weight.
Statistical analyses
Continuous variables are presented as mean ± standard deviation. To compare the variables between the two groups, the unpaired Student’s t test was used. The effects of leukocyte filtration were tested using the paired t test.
Animal care
All animals received humane care in compliance with the "Principles of Laboratory Animal Care" formulated by the National Society for Medical Research and the "Guide for the Care and Use of Laboratory Animals" prepared at the National Academy of Sciences and published by the National Institutes of Health (NIH publication 86-23, revised 1985).
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Results
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The leukocyte count in group 2 fell from 9.5 ± 3.5 x 103 to 0.4 ± 0.3 x 103/mm3 after filtration (Sepacell, Asahi Medical Co, Ltd, Tokyo, Japan). The average efficacy of leukocyte filtration was approximately 96.0% ± 2.3% (Fig 2). The mean grades of tissue ICAM-1 at 3 and 4 hours after the onset of reperfusion were significantly higher in group 1: p = 0.0047 at 3 hours and p = 0.0014 at 4 hours (Fig 3). These results were well correlated with myocardial function. The mean stroke work indices at 2, 3, and 4 hours after the onset of reperfusion were significantly higher in group 2: p = 0.0498 at 2 hours, p = 0.0016 at 3 hours, and p = 0.0024 at 4 hours (Fig 4). The myocardial water contents were not different between groups. The percent wet/dry ratio was 82.0% ± 3.0% in group 1 and 81.1% ± 4.1% in group 2 (p > 0.05) (Figs 5, 6).
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Fig 4. The mean grades of tissue ICAM-1 expression in the myocardium, were significantly higher in the control group (group 1) than the leukocyte-depleted group (group 2) at 3 hours (p = 0.047) and 4 hours (p = 0.0014) after the onset of cardiac reperfusion.
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Fig 5. The mean stroke work indices were significantly greater in the leukocyte-depleted group (group 2) than in the control group (group 1) at 2, 3, and 4 hours after the onset of reperfusion (p = 0.0498, p = 0.0016, and p = 0.0024, respectively).
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Fig 6. The myocardial water content, expressed as percent wet–dry ratio, was not significantly different between the two groups (LD = leukocyte-depleted group).
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Comment
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Ischemia-reperfusion injury in heart transplantation affects both early graft function and late graft survival. It has been demonstrated that leukocytes, in addition to the endothelium, play a key role in this injury [5–7]. The adhesive interactions between circulating leukocytes and the endothelium are crucial steps in such serial inflammatory reactions, which are modulated by adhesion glycoproteins. In our study the attenuating effect of leukocyte-depleted reperfusion, after a period of cold ischemic storage, on the myocardial expression of ICAM-1 as well as its relationship to functional recovery was demonstrated in the isolated piglet working heart perfusion model.
In vivo animal models have demonstrated that the inhibition of neutrophil accumulation by an antiinflammatory agent or by systemic leukocyte depletion reduces the infarct size [6]. On reperfusion, neutrophils promptly infiltrates the previously ischemic tissue and the neutrophil infiltration progresses with the length of reperfusion [8]. Leukocytes, in part by their large size and deformability, plug myocardial capillaries during reperfusion [9, 10]. Once activated, neutrophils engage several mechanisms that are responsible for reperfusion injury, including the production of oxygen free radicals and lipid peroxydation [11]. The release of other chemotactic substances further results in the migration and activation of neutrophils and macrophages, resulting in an amplification of the inflammatory response [12]. Molecular mechanisms underlying leukocyte movements from the circulation into the tissues consist of initial rolling, firm adhesion, and transmigration through the expression of the endothelial adhesion molecules such as P-selectin, ICAM-1, and E-selectin. In vitro studies have demonstrated that stimulated expression of endothelial P-selectin is very rapid (ie, within 10 minutes) but that the induction of ICAM-1 and E-selectin requires several hours for protein synthesis [3, 10]. Weyrich and colleagues [1] demonstrated that maximal expression of P-selectin occurred 20 minutes after reperfusion and was sustained for at least 270 minutes in an in vivo feline ischemia-reperfusion model. However, constitutive ICAM-1 expression was seen throughout the vasculature, and these levels increased five-fold at 150 and 270 minutes after reperfusion. Our results were in accordance with these findings. In our group 1, ICAM-1 expression was seen at all time points, and its intensity increased significantly 3 and 4 hours after the onset of reperfusion. These observations suggest that constitutive ICAM-1 play a role in leukocyte adhesion to endothelial cells during early reperfusion. However its effect appears to be enhanced with longer periods of reperfusion, as its expression increased over time. Reperfusion of ischemic myocardium undoubtedly releases the humoral mediators that induce high levels of ICAM-1 on the endothelial surface. Previous study demonstrated that neutrophil-derived TNF-
, peroxides, and other inflammatory mediators cause marked up-regulation of ICAM-1 expression on their cell surfaces [13]. Meanwhile, the delayed expression of ICAM-1 on the vascular endothelium, with its subsequent tight adhesion with leukocytes and their accumulation, results in inflammatory responses and aggravates myocardial cell death [2]. In our group 2, the expression of ICAM-1 was also observed at all time points, but there was no significant increase even at 3 and 4 hours after the onset of reperfusion. Therefore, we suggest that cardioprotective and endothelium-preserving effects can be expected in the ischemic-stored myocardium reperfused with leukocyte-depleted blood. Breda and colleagues [14] proved the functional and ultrastructural benefits of leukocyte depletion in their study of 12-hour ischemia and 4-hour reperfusion using an isolated working heart perfusion model. Our results confirmed these hypotheses in that stroke work indices were better preserved in the leukocyte-depleted group at 3 and 4 hours after reperfusion. Thus, it is not surprising that a strategy inhibiting the activity of adhesion molecules like ICAM-1 can be a good option to protect myocardium. Numerous interventions aimed at preventing the adhesion and activation of leukocytes have proven to attenuate reperfusion injury [6, 7, 14–18]. In our group 2 of the present study, the heart was reperfused with leukocyte-depleted whole blood during the entire length of the study period. This allowed us to see the uniform effect of leukocyte depletion on the adhesion molecule expression. In addition to ischemic-reperfusion injury, cardiopulmonary bypass itself may cause a whole-body inflammatory response with the leukocyte–endothelial interaction in other organs. Our reperfusion circuit, comprised of a membrane oxygenator and roller pumps, is thought to be a suitable model to test the sole effect of leukocyte-depleted reperfusion on the myocardial expression of adhesion molecules by excluding the reaction of other parts of the body. However, in our study, 4 hours of reperfusion time might not be long enough to define the effect of leukocyte depletion on the expression of ICAM-1, considering the time required in protein synthesis at the level of myocardium.
In conclusion, our study using an isolated piglet working heart perfusion model demonstrated that leukocytes have a significant effect on the expression of ICAM-1 in the reperfused myocardium, and that time-dependent functional deterioration of the myocardium is well correlated with the degree of ICAM-1 expression.
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Acknowledgments
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We thank Chul Jun Seok, Hyang Min Jung, and Eul Kyung Kim for their assistance in the laboratory. This study was supported by grant 03-97-061 from the Seoul National University Research Fund.
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References
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Accepted for publication April 25, 2000.
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Abstract
Introduction
