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Ann Thorac Surg 2005;79:204-211
© 2005 The Society of Thoracic Surgeons

Original article: Cardiovascular

Changes in P-Selectin Expression on Cardiac Microvessels in Blood-Perfused Rat Hearts Subjected to Ischemia-Reperfusion

Andrew O. Chukwuemeka, FRCSa, K. Alun Brown, PhDb, Graham E. Venn, MSa, David J. Chambers, PhDa,*

a Cardiac Surgical Research/Cardiothoracic Surgery, Rayne Institute, Guy's and St. Thomas' NHS Trust, St. Thomas' Campus
b Department of Immunobiology, GKT School of Medicine, King's College London, Guy's Campus, London, United Kingdom

Accepted for publication June 25, 2004.

* Address reprint requests to Dr Chambers, Cardiac Surgical Research, The Rayne Institute, St. Thomas' Hospital, London SE1 7EH, UK (E-mail: david.chambers{at}kcl.ac.uk).


   Abstract
Top
 Abstract
Introduction
 Material and Methods
 Results
 Comment
 Acknowledgments
 References
 
BACKGROUND: During cardiac surgery involving cardiopulmonary bypass, activation of polymorphonuclear cells is believed to contribute to ischemia-reperfusion injury and subsequent myocardial impairment of function. The early tethering of polymorphonuclear cells to blood vessel walls depends upon recognition of the adhesion molecule P-selectin on endothelium. The purpose of this study was to define the kinetic changes in expression of P-selectin on myocardial vessels in a model of global ischemia-reperfusion injury.

METHODS: In a novel recirculating blood-based perfusion system, rat hearts were subjected to 30 minutes of aerobic perfusion, 60 minutes of global ischemia, and 60 minutes of reperfusion, or to 120 minutes of continuous aerobic blood perfusion (with or without leukocyte/platelet depletion). Heart function (left ventricular developed pressure), heart rate, and perfusion pressure were monitored throughout. Hearts were sampled at defined periods for microvascular expression of P-selectin, identified by immunohistochemistry.

RESULTS: In control (nonperfused) hearts and in hearts subjected to perfusion and ischemia, few cardiac vessels (8% to 16%) expressed P-selectin. After 15 minutes of reperfusion, P-selectin was present on the majority of vessels (77%; p < 0.05) but expression decreased subsequently throughout the remaining duration of reperfusion. Interestingly, upregulation of P-selectin also occurred when hearts were subjected to continuous perfusion alone (no ischemia), but this upregulation was less rapid. Depletion of leukocytes/platelets from the blood perfusate did not modify P-selectin expression.

CONCLUSIONS: The augmented expression of P-selectin on myocardial vessels during reperfusion of ischemic hearts probably reflects changes induced during global ischemia and by the duration of perfusion through the nonbiological tubing of the circuit. That is likely to mimic the effects initiated during cardiopulmonary bypass.


   Introduction
Top
 Abstract
 Introduction
Material and Methods
 Results
 Comment
 Acknowledgments
 References
 
Myocardial damage arising from ischemia-reperfusion remains a major complication of cardiac surgery involving cardiopulmonary bypass (CPB). Activation of polymorphonuclear cells (PMNs) and inflammatory factors (such as complement and cytokines) by the nonbiological circuitry of the CPB is thought to contribute to vascular damage and an impairment of myocardial function [1]. Ischemia-reperfusion injury is characterized by the attachment of large numbers of PMNs to the endothelial cells lining vessel walls in the heart [2]. This excessive interaction could induce cardiac damage either by disruption of blood vessel walls so as to increase vascular permeability [3] or by promoting the tissue infiltration of PMNs and their extracellular release of oxyradicals and lysosomal enzymes [4]. Binding of PMNs to endothelium involves adhesion molecules on both cell types interacting with corresponding ligands [5]. At sites of inflammation, the early tethering or rolling of PMNs along blood vessel walls is dependent upon cells recognizing the vascular ligand P-selectin on the endothelial surface by recognition of P-selectin glycoprotein ligand-1 [6]. Without this initial attachment, additional neutrophil receptors (eg, CD11b) are unlikely to effect firm adhesion by binding to other vascular ligands such as intercellular adhesion molecule-1 [5]. During CPB, the expression of both P-selectin glycoprotein ligand-1 and CD11b are increased on activated PMNs [2]. Potential inducers of this upregulation include cytokines [7, 8] and the complement peptide C5a, which is generated by contact of blood with foreign surfaces [9].

Resting endothelial cells express little or no P-selectin, but within minutes of cellular activation by inflammatory stimuli such as histamine, thrombin, and H2O2, there is a marked upregulation of P-selectin due to its rapid translocation from cytoplasmic Weibel-Palade bodies [10]. The importance of P-selectin to PMN attachment is illustrated by studies whereby antagonizing its expression suppresses the acute inflammation of the Arthus reaction [11] and alleviates reperfusion injury of the rabbit ear [12]. In a number of species, anti-P-selectin monoclonal antibodies reduced infarct size in isolated hearts subjected to regional ischemia [13, 14], and a soluble P-selectin glycoprotein ligand limited ischemia-reperfusion injury in rat kidney grafts [15]. Whether antagonizing P-selectin expression attenuates the myocardial injury induced during global ischemia-reperfusion remains a matter of conjecture as there is no information concerning the distribution and kinetics of expression of P-selectin during this inflammatory disorder. To address this question, the purpose of the current study was to use a novel model of global ischemia-reperfusion injury incorporating continuous blood perfusion of isolated rat hearts that would allow examination of changes in P-selectin expression on cardiac microvessels.


   Material and Methods
Top
 Abstract
 Introduction
 Material and Methods
Results
 Comment
 Acknowledgments
 References
 
Animals
Isolated hearts and blood samples were obtained from adult male Wistar rats of 300 g to 330 g body weight (Bantin and Kingman, Hull, UK). All animals received humane care in compliance with the "Guide for the Care and Use of Laboratory Animals" published by the U.S. National Institutes of Health (NIH Publication No. 85-23, revised 1996), and with the "Guidance on the Operation of the Animals (Scientific Procedures) Act 1986" published by Her Majesty's Stationery Office, London, England.

Recirculating Blood Perfusion System
A recirculating blood perfusion circuit was developed with a basic design that resembled a clinical CPB circuit (Fig 1). The circuit consisted of a plastic reservoir inserted into a temperature-controlled (37°C) heart chamber; this reservoir contained approximately 5 mL of the blood perfusate. The base of the reservoir was lined with a 200 µm nylon gauze (Cadisch Precision Meshes, London, UK) to prevent any particulate debris (eg, epicardial fat, aggregates) from entering the circuit tubing. A length of silicon tubing (Altesil; Altec, Alton, Hampshire, UK), 2.5 mm bore, 1.0 mm wall thickness, passed from the base of the reservoir through a peristaltic roller pump (Gilson Minipuls 3; Anachem, Luton, Bedfordshire, UK) and into a home-made "membrane" oxygenator. The oxygenator consisted of a coil (of approximately 30 turns) of thin-walled, gas-permeable silicon tubing (OsteoTec Ltd, Christchurch, Dorset, UK), 1.47 mm bore, 0.46 mm wall thickness, which was housed in a temperature-controlled (37°C) glass chamber with a lower inlet into which a gas mixture of 95% O2:5% CO2 was passed in countercurrent to the perfusate through the tubing. The gas mixture escaped at the top of the chamber around the sides of a loose-fitting silicon bung; this approach ensured that the perfusate maintained an O2 tension around 150 mm Hg (148 ± 7 mm Hg at 15 minutes and 134 ± 24 mm Hg at 120 minutes of continuous perfusion). The 5% CO2 content of the gas mixture gave a pCO2 of 35 ± 2 and 35 ± 3 mm Hg at 15 and 120 minutes of perfusion, respectively, and maintained perfusate pH within the physiologic range (at pH 7.4 throughout perfusion). The oxygenator was connected, through silicon tubing (Altesil), to a water-jacketed temperature-controlled (37°C) aortic cannula, through which the perfusate returned to the reservoir. When global ischemia was induced, by closing flow to the aortic cannula using a three-way tap, the circuit reservoir was moved aside and the blood perfusate flow diverted directly into the reservoir to avoid any stasis of the blood. At the same time, a separate temperature-controlled heart chamber maintained the heart at 37°C (see Fig 1). The total volume of the perfusate circuit was approximately 16 mL.



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  		Fig 1. The recirculating Langendorff blood perfusion circuit. A plastic reservoir was inserted into a temperature-controlled heart chamber; tubing from the chamber passed through a peristaltic pump and into a home-made oxygenator consisting of coils of thin-walled, gas-permeable silicon tubing. The oxygenator was encased in a temperature-controlled glass chamber through which 95% O2:5% CO2 was passed countercurrent to the flow of the perfusate. It was connected to a temperature-controlled aortic cannula which returned the perfusate to the reservoir (for further details, see Methods). During ischemia, a three-way tap before the aortic cannula diverted the perfusate directly into the reservoir and maintains continuous circulation of the perfusate. The heart was sealed within a separate chamber and the temperature maintained at 37°C.

 
At the end of each experiment, the circuit was thoroughly washed with 5 L cold and 5 L boiling deionized water.

Perfusion Solutions
PREPARATION OF BLOOD-BASED PERFUSATE
All perfusates were analyzed using an automated blood-gas analyser (Stat Profile 5; NOVA Biomedical, Waltham, MA), and their composition is shown in Table 1. The extracorporeal perfusion circuit was primed with modified Krebs-Henseleit buffer solution (MKH) and Gelofusine (G), a modified fluid gelatin in saline, mixed in a ratio of 1:1 to give a MKH/G solution (Table 1).


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  		Table 1. Composition of Perfusates Applied to Isolated Rat Heart Preparations
 
Rats, anesthetized by intraperitoneal injection of pentobarbitone (2 mL/kg of a 60 mg/mL solution) and anticoagulated with heparin (1000 IU/kg), were exsanguinated by withdrawal of blood from the inferior vena cava. This blood was added to the MKH/G solution prime in the perfusion circuit to give a 1:1 mixture (Table 1) and was continuously circulated for 2 minutes (at a flow rate of 10 mL/min) to ensure adequate mixing between the MKH/G solution and the rat blood. This blood-based perfusate (with a hemoglobin of approximately 6.5 g/dL) was oxygenated through the membrane oxygenator incorporated in the perfusion circuit, and the 37°C perfusate flow rate was maintained at 4.5 mL/min by a Gilson Minipuls 3 peristaltic pump.

LEUKOCYTE-DEPLETED BLOOD-BASED PERFUSATE
Blood was obtained by venepuncture of the inferior vena cava of an anesthetized rat as described above, and centrifuged at 1,000g for 10 minutes. The buffy coat layer, containing leukocytes and platelets, was removed by aspiration, and the red blood cells in the pellet resuspended in the plasma by gentle manual agitation. The leukocytes and platelets were enumerated by an automated cell counter (Gen-S System 2 Automated Cell Counter; Beckman-Coulter, Miami, FL). Preliminary experiments with 5 rats demonstrated that the above procedure reduced the mean number of leukocytes from 4.1 ± 10.4 x 109/L to 1.3 ± 0.3 x 109/L (68% decrease; p < 0.001) and the mean number of platelets from 725 ± 40 x 109/L to 246 ± 33 x 109/L (64% decrease; p < 0.001). Subsequently, the blood, depleted of leukocytes and platelets, was introduced into the perfusion circuit in exactly the same manner as described earlier.

Heart Perfusion
Rats were anesthetized by intraperitoneal injection of pentobarbitone (2 mL/kg of a 60 mg/mL solution), and anticoagulated with intravenous administration of 1,000 IU/kg heparin. Hearts were rapidly excised and immersed in cold (4°C) Gelofusine; the aorta was immediately cannulated and hearts were perfused at a constant flow rate of 4.5 mL/min in the Langendorff mode (retrograde perfusion through the aorta that forces the aortic valve closed and perfuses the myocardium antegradely through the coronary arteries [16]). After removal of the left atrial appendage, a fluid-filled balloon catheter (constructed from cling film), attached to a pressure transducer, was introduced into the left ventricle through the mitral valve. The balloon was gradually inflated with water until a left ventricular end-diastolic pressure of 5 to 10 mm Hg was obtained. During perfusion, heart function (left ventricular systolic pressure, heart rate, and left ventricular end-diastolic pressure) was measured. Left ventricular developed pressure was calculated as left ventricular systolic pressure minus left ventricular end-diastolic pressure. A second pressure transducer attached to a sidearm of the aortic cannula allowed the measurement of perfusion pressure. The output from these pressure transducers was recorded using a MacLab 8s analogue:digital converter connected to a PowerMacintosh computer, employing MacLab Chart software (AD Instruments Ltd, Hastings, UK). Preliminary experiments showed that initial perfusion pressure was 69 ± 9 mm Hg (at 15 minutes of perfusion) which gradually increased to 91 ± 13 mm Hg after 120 minutes of perfusion. Left ventricular developed pressure gradually decreased during 120 minutes of perfusion from an initial value of 174 ± 7 mm Hg to 144 ± 13 mm Hg; this was an acceptable rate of decline for a Langendorff preparation [16].

Perfusion Protocols
Isolated rat hearts, perfused in the Langendorff mode, were subjected to 30 minutes of aerobic perfusion, 60 minutes of global ischemia, and 60 minutes of reperfusion. Hearts were sampled at predetermined time points (n = 3 hearts/time point) throughout the protocol by perfusion fixation with 10% formalin for 30 seconds. They were then cut transversely into three segments of approximately equal size and stored in 10% formalin for 24 hours. Control hearts were perfusion fixed with 10% formalin immediately after removal from the animal. Hearts were sampled at 0 and 30 minutes of aerobic perfusion, 30 and 60 minutes of global ischemia, and 15, 30, 45, and 60 minutes of reperfusion.

In addition, separate groups of hearts were continuously aerobically perfused (with blood-based perfusate) for up to 120 minutes and sampled by perfusion fixation at 0, 30, 45, 60, 90, and 120 minutes (n = 3 hearts/time point). Similarly, hearts were continuously aerobically perfused with leukocyte-depleted blood-based perfusate for up to 120 minutes and sampled by perfusion fixation at 0, 30, 60, 90, and 120 minutes (n = 3 hearts/time point).

Antibodies
A polyclonal rabbit antirat CD62P (P-selectin) antibody was obtained from PharMingen, San Diego, California. The ABC detection system (Vector Laboratories, Burlinghame, CA) was used with a biotinylated secondary donkey antirabbit antibody (Jackson ImmunoResearch Laboratories, West Grove, PA).

Staining for P-selectin Expression
After fixation, heart segments were processed through graded ethanol (70%, 90%, and 100%) and xylene before being embedded in paraffin wax. Serial sections (5 µm) were prepared with a Leitz rotary microtome (Leica; Milton Keynes, Bedfordshire, UK) and mounted onto glass slides coated with Vectabond (Vector Laboratories, Burlinghame, CA).

Tissue sections were dewaxed by immersion in xylene for 5 minutes and rehydrated through graded ethanol (5 minutes in each of 100%, 90%, 70%, and 50%). Endogenous peroxidase activity was attenuated by immersing sections for 5 minutes in a solution of hydrogen peroxide in methanol (2.5% H2O2 by volume in 74 OP methanol). The sections were washed in distilled water for 5 minutes, and incubated with trypsin (0.2 mL in 3.8 mL of 0.1% CaCl2) for 10 minutes at 37°C for antigen retrieval, treated with DAKO Universal Block (0.25% Casein in phosphate-buffered saline; DAKO Corporation, Carpinteria, CA) for 30 minutes to prevent nonspecific binding of primary antibodies and incubated overnight (at 4°C) with 3 µg/mL of the anti-P-selectin antibody. Bound primary antibodies were detected by a 45-minute incubation with a bridging antibody followed by a further incubation for 30 minutes with a 1:20 dilution of avidin peroxidase-antiavidin peroxidase complex (ABC detection system; Vector Laboratories) [17]. The sections were then counter-stained by the application of Nuclear Fast Red dye (Vector Laboratories) for 6 minutes, dehydrated and mounted. Blood vessels staining for P-selectin were visualized by light microscopy (x40 magnification). At least two sections were examined from each of the three segments from each heart (3 hearts/time point); thus, a total of at least 18 sections were measured for each time point. The number of vessels stained for P-selectin were expressed as a percentage of the total vessels.

Statistics
Statistical analysis was performed by employing a commercially available statistical package (Statview; SAS Institute, Cary, NC). Data were analyzed using one-way analysis of variance, and where this revealed a significant difference, the Student-Newman-Keuls test was used to identify the difference posthoc. All data are reported as mean ± standard error of the mean (SEM). A p value of less than 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Material and Methods
 Results
Comment
 Acknowledgments
 References
 
P-Selectin Expression
ISCHEMIA-REPERFUSION
In control (nonperfused) hearts, the majority of microvessels were devoid of P-selectin, in contrast to the high preponderance of microvessels bearing this molecule in hearts subjected to ischemia and reperfusion (Fig 2). A more detailed examination revealed that only 8% ± 1% of vessels in nonperfused hearts showed positive staining by the anti-P-selectin antibody (Fig 3). During 30 minutes of perfusion, there was a slight but nonsignificant increase in the number of vessels expressing P-selectin. When the heart was subjected to 60 minutes of ischemia, there was no upregulation of P-selectin expression. In contrast, after 15 minutes of reperfusion, the majority of vessels were found to express P-selectin (77% ± 2%; p < 0.05); thereafter, there was a progressive decline in P-selectin expression, from 63% ± 5% at 30 minutes to 36% ± 2% at 60 minutes of reperfusion (Fig 3).



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  		Fig 2. Expression of P-selectin on cardiac microvessels. Sections of myocardial tissue were treated with a primary antibody against P-selectin and the staining developed by the avidin-antiavidin peroxidase complex. Panels A and B show blood vessels in normal heart tissue do not express P-selectin; panels C and D demonstrate the marked expression of P-selectin (indicated by arrows) on the luminal walls of blood vessels in hearts subjected to 15 minutes of reperfusion after ischemia. (Panels A and C: magnification x100; panels B and D: magnification x400.)

 


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  		Fig 3. Upregulation of P-selectin on blood vessel walls during perfusion, ischemia, and reperfusion. After 30 minutes of control perfusion with modified Krebs-Henseleit buffer solution with Gelofusine (MKH/G) plus blood at a flow rate of 4.5 mL/min, isolated rat hearts were subjected to 60 minutes of ischemia (shaded area) and 60 minutes of reperfusion. Hearts were sampled throughout the experimental duration and processed for immunohistochemical staining. Results are expressed as the percentage of vessels expressing P-selectin. Values are mean ± SEM of at least 18 measurements per time point (3 hearts, each cut into 3 segments and at least 2 sections per segment). *p less than 0.05 compared with 30-minute value.

 
CONTINUOUS PERFUSION
To investigate whether the rapid upregulation of P-selectin (Fig 3) was due entirely to the process of reperfusion or to whether a nonspecific effect due to the nonbiological circuitry was involved, P-selectin expression on cardiac vessels was examined during continuous perfusion in the absence of ischemia and reperfusion. Figure 4 shows that there was little modification of P-selectin during the initial 30 minutes of perfusion with the MKH/G plus blood, but this increased to 46% ± 3% at 45 minutes and to 68% ± 3% after a further 15 minutes (60 minutes); this level was maintained at 90 minutes but decreased to 50% ± 3% at 120 minutes of perfusion.



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  		Fig 4. Upregulation of P-selectin on blood vessel walls during continuous perfusion. Isolated rat hearts were perfused with either modified Krebs-Henseleit buffer solution with Gelofusine (MKH/G) plus blood (solid line) or MKH/G plus blood depleted of leukocytes and platelets (dashed line) at a flow rate of 4.5 mL/min for 120 minutes. Hearts were sampled throughout the experimental duration and processed for immunohistochemical staining. Results are expressed as the percentage of vessels expressing P-selectin. Values are mean ± SEM of at least 18 measurements per time point (3 hearts, each cut into 3 segments and at least 2 sections per segment). *p less than 0.05 compared with 0-minute value.

 
To determine whether leukocytes and platelets may have contributed to P-selectin upregulation, experiments were undertaken with a perfusate solution depleted of leukocytes and platelets. It is apparent (Fig 4) that changes in P-selectin expression were similar to those induced by the nondepleted perfusate. There was, however, a tendency for an increased expression at 30 minutes and a decreased expression at 90 minutes compared with the normal (nondepleted) perfusate solution, but these differences were not significant.


   Comment
Top
 Abstract
 Introduction
 Material and Methods
 Results
 Comment
Acknowledgments
 References
 
In this study, a novel blood-perfused model of CPB with isolated rat hearts was used to determine the time-related responses of P-selectin expression during global ischemia and reperfusion. We observed that P-selectin expression, which is normally present on a minority of cardiac vessels, was markedly and rapidly upregulated after 15 minutes of reperfusion but gradually decreased thereafter over the remaining reperfusion period. The upregulation of P-selectin was also induced by continuous perfusion; this effect does not appear to be dependent on the presence of leukocytes or platelets in the blood perfusate.

The model of CPB used in this study was specifically developed to include a number of features associated with the clinical situation. An important aspect of the model was that the circuit was made sufficiently small so as to incorporate rat blood (taken from a single donor animal), in preference to other systems that have used washed red cells from different species [16, 18, 19]. As a consequence, the perfusate was continuously recirculated and the rat blood was diluted to the same degree as occurs clinically during CPB, such that a hematocrit of approximately 20% was achieved. In addition, we incorporated a membrane oxygenator to minimize any potential disturbance to the blood during oxygenation, and a roller pump for circulation of the perfusate.

The cardiovascular dysfunction associated with ischemia and reperfusion may arise from an excessive binding to myocardial vessels of PMNs, which subsequently release inflammatory factors such as oxygen radicals that damage endothelium and the underlying tissue [20]. Supporting this view is the demonstration that, in isolated rat hearts, oxygen radicals only appear in the coronary artery when neutrophils are present in the perfusate [21], and that antagonizing PMN activation attenuates myocardial injury [22–24]. Other studies have also demonstrated that PMN depletion prevents postischemic contractile dysfunction [25] and impairs the phenomenon of "no-reflow" [26, 27], which occurs when myocardial capillary beds fail to reperfuse after restoration of blood flow in the presence of normal perfusion pressure.

We chose P-selectin for study because its expression on endothelial cells is purported to have a major role in the tethering and rolling of PMNs along the endothelium, a process that precedes the firm adhesion of the cells to blood vessel walls [5]. The importance of this molecule to the inflammatory effects of regional ischemia-reperfusion injury is illustrated by experimental studies in canine and feline hearts subjected to regional occlusion of the left anterior descending coronary artery. Administration of anti-P-selectin antibodies at the time of reperfusion reduces infarct size, impairs PMN adhesion to coronary endothelium, and enhances endothelial preservation [13, 28, 29]. Anti-P-selectin antibodies also attenuate the no-reflow phenomenon and myocardial necrosis and inhibit PMN-mediated systolic and diastolic function [30]. The major ligand for P-selectin on the PMN surface is P-selectin glycoprotein ligand-1, and peptide analogues of this ligand protect against infarction due to coronary occlusion and attenuate PMN extravasation in ischemia-reperfusion injury in vivo [31] and in vitro [32]. Moreover, mice deficient in P-selectin are resistant to coronary ischemia-reperfusion injury [33], whereas P-selectin expressed on activated platelets augment PMN-endothelial cell adherence [34].

In the present study, P-selectin was confined to a minority of cardiac microvessels in normal rat hearts, and this expression showed little change during either the initial 30 minutes of perfusion or the 60 minutes of ischemia. However, after 15 minutes of reperfusion, P-selectin was present on the majority of vessels and extending the reperfusion time produced a gradual decline in P-selectin expression. The kinetics of this P-selectin response to global ischemia-reperfusion injury are similar to those described for an upregulation of P-selectin during regional ischemia-reperfusion injury [35], although in a chronic murine model of myocardial reperfusion injury, the peak expression of P-selectin was recorded 24 hours after reperfusion [36]. A rapid upregulation of P-selectin expression is due to its translocation from a preformed pool within the cytoplasmic Weibel-Palade bodies of the endothelial cells whereas the loss of expression is probably due to enzymatic cleavage or endocytosis [10, 37]. The expression of P-selectin has previously been measured in rat hearts by Lefer and colleagues [22] but only at the end of an ischemia/reperfusion protocol. They showed no P-selectin expression in the absence of PMNs and platelets (in crystalloid perfused hearts), but the presence of a combination of these blood components significantly enhanced P-selectin expression and this could be attenuated by the antiselectin agent sialyl Lewisx oligosaccharide. Interestingly, the levels of P-selectin expression that were observed in this study [22] tend to be considerably lower than those of our study, and this may relate to the time at which their hearts were sampled (at a time that we have shown that P-selectin expression was decreasing) and to perfusion with a crystalloid solution rather than a blood-based solution.

An interesting and unexpected finding in our study was the observation that P-selectin expression was also upregulated when isolated hearts were subjected to just continuous perfusion with the blood-based perfusate. These findings suggest that induction of P-selectin may not only be a feature of ischemia and reperfusion (where reperfusion appears to act as a rapid trigger for the P-selectin upregulation), but a more general consequence of continuous perfusion through the nonbiological tubing of the extracorporeal circuit. It has previously been shown [22] that rat hearts perfused with a nonrecirculating crystalloid perfusate (containing no blood components) had no evidence of any P-selectin expression.

The upregulation of P-selectin probably relates to the trapping of PMNs in coronary capillaries that is reported to occur within 5 minutes of reperfusion [35]. During CPB, passage of blood through the nonbiological tubing leads to PMN activation and generation of the respiratory burst [38, 39]. The possibility that induction of P-selectin expression arises from the release of oxidant species from activated PMNs is not wholly supported by our data, since depleting leukocytes and platelets from the perfusate failed to modify the kinetics of P-selectin expression. However, the depletion technique used in this study only removed two thirds of the leukocytes (and platelets) from the perfusate, and it is conceivable that sufficient of these blood components remained to participate in the upregulation of P-selectin expression. Another likely consideration is that the increased expression of P-selectin is attributable to the activity of other known agonists such as thrombin and histamine [40].

In conclusion, our study suggests that the induction of global ischemia-reperfusion may not be the only means by which P-selectin expression is increased. The demonstration that subjecting cardiac vessels to a continuous recirculation of blood perfusate through an extracorporeal circuit also upregulates P-selectin implies that additional mechanisms may be contributing to this induction of P-selectin expression during CPB.


   Acknowledgments
Top
 Abstract
 Introduction
 Material and Methods
 Results
 Comment
 Acknowledgments
References
 
These studies were funded by a Studentship Grant from The Garfield Weston Trust to Mr Chukwuemeka.


   References
Top
 Abstract
 Introduction
 Material and Methods
 Results
 Comment
 Acknowledgments
 References
 

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