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*Department of Anesthesiology, Advocate Illinois Masonic Medical Center, and Department of Anesthesiology, University of Illinois College of Medicine; Department of Physiology and Biophysics, University of Illinois College of Medicine, Chicago, Illinois
Address correspondence and reprint requests to George J. Crystal, PhD, Department of Anesthesiology, Advocate IL Masonic Medical Center, 836 West Wellington Avenue, Chicago, IL 60657–5193. Address e-mail to gcrystal{at}uic.edu.
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Abstract |
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Introduction |
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Volatile anesthetics have been demonstrated to protect the myocardium against ischemic-reperfusion injury (8), but despite extensive investigation, the mechanisms underlying this effect remain unclear. Activation of protective signaling pathways within the myocytes involving the adenosine triphosphate-sensitive potassium (KATP) channels appears to play a prominent role (8), but various lines of evidence have suggested that inhibitory actions on inflammatory pathways may also be involved. First, isoflurane reduced neutrophil adherence to the endothelium and the cardiac and endothelial dysfunction caused by the activated neutrophils (9–12). Furthermore, isoflurane reduced the cytokine-induced death of cultured endothelial and smooth muscle cells and the release of tumor necrosis factor-
(13).
Although some studies have shown that isoflurane has no effect on platelet function (14–16), others have suggested that it has an inhibitory effect at clinically relevant concentrations (17–20). In addition, several reports have demonstrated that volatile anesthetics can modulate the formation of platelet-neutrophil aggregates and expression of P-selectin on platelets but that these effects may differ among the various anesthetics (21–23). No study has assessed whether isoflurane (or any other volatile anesthetic) can affect the ability of platelets to modulate neutrophil-induced coronary endothelial dysfunction. Such information is important in gaining a more complete understanding of the mechanisms by which volatile anesthetics protect the myocardium from reperfusion injury in vivo.
The first objective of this study was to demonstrate the ability of platelets to enhance neutrophil-induced coronary endothelial dysfunction. After this was accomplished, we evaluated the effect of isoflurane on this phenomenon. Mechanistic insights were obtained by evaluating the associated changes in superoxide production and vascular adherence of the activated neutrophils.
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Methods |
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Experiments were performed on 25 healthy, heartworm-free mongrel dogs of either sex (weight, 18–26 kg). The dogs were anesthetized with an IV bolus injection of 30 mg/kg sodium pentobarbital and mechanically ventilated (Air Shields Inc, Hatboro, PA) with oxygen-enriched room air. The right carotid artery was cannulated to permit withdrawal of blood for separation of neutrophils and platelets.
Neutrophils were separated as described previously (11). Arterial blood was collected in tubes and anticoagulated with 1.6% citric acid and 2.5% sodium citrate (pH 5.4) in 10 mL of 6% dextran solution in buffered Hanks’ balanced salt solution (HBSS). The blood was maintained at room temperature while erythrocytes sedimented (approximately 40 min). The leukocyte-rich plasma layer was carefully aspirated and centrifuged at 500g at 4°C for 10 min. Contaminating erythrocytes in the pellet were removed by hypotonic lysis for 20 s with 9 mL of sterile distilled water. Subsequent addition of 3 mL of 0.6 M KCl and 15 mL of buffered HBSS rapidly returned the cells to isotonicity. The leukocyte-rich suspension was centrifuged at 500g at 4°C for 10 min, after which time the cells were resuspended in 2 mL of HBSS, layered on the top of 3 mL of Ficoll-Pacque, and centrifuged again at 800g at 4°C for 20 min. The resulting pellet was rinsed with HBSS. The neutrophils were resuspended in HBSS in preparation before experimental use. Our procedure for neutrophil isolation yields neutrophil suspensions that are 98% pure and more than 95% viable as evaluated by Wright’s staining and trypan blue exclusion (11,24).
Platelets were isolated by centrifugation as described previously (25). Briefly, arterial blood was collected in tubes and combined with citrate anticoagulant A to yield final concentrations of 9.3 mM sodium citrate, 0.7 mM citric acid, and 14 mM dextrose. This blood was then centrifuged at 55g at 4°C for 40 min. The platelet-rich plasma layer was carefully collected and added into an equal volume of cold citrate anticoagulant B solution (in mM: sodium citrate 93, citric acid 7, dextrose 105, and KCl 5, pH 6.5), and then the mixture was centrifuged at 600g at 4°C for 20 min. The supernatant was discarded, and the resulting platelet pellet was resuspended in a small volume of the citrate anticoagulant B. A platelet count of this suspension was obtained manually after Wright’s staining (24).
To quantify neutrophil-induced damage to the endothelium, dogs were killed with an overdose of IV pentobarbital sodium (80 mg/kg), and the heart was rapidly excised and immediately placed into cold (4°C), oxygenated Krebs solution with the following composition (mM): NaCl 118, KCl 4.75, MgSO4 1.19, KH2PO4 1.12, CaCl2 2.54, NaHCO3 12.5, glucose 10. The left anterior descending and circumflex coronary arteries were carefully dissected, cleaned of connective and adipose tissue without disturbing the endothelium, and cut into rings 2–3 mm in length as described previously (11). The rings were mounted on stainless steel hooks, placed into organ baths containing 10 mL of Krebs solution aerated with a gas mixture of 95%O2–5% CO2 at 37°C, and connected to isometric force transducers (Radnoti Model TR 001). Changes in isometric force were digitized at 3 Hz using an analog-to-digital converter and analyzed using a videographic program (SPECTRUM®; Triton Technology, San Diego, CA). The rings were initially stretched to yield a preload of 1.0 g of tension. After equilibration for 20 min, the contraction response to KCl (30 mM) was determined as tension was increased in 1.0-g steps until the maximal response was observed. This prestretch tension was used in subsequent procedures. The rings were then thoroughly washed and the optimal contracting dose for U46619 (Upjohn, Kalamazoo, MI), a thromboxane A2 mimetic agent, was determined. A concentration-response curve for U46619 was generated for each ring using concentrations over the range 2.5 to 5.0 nM. The concentration for U46619 that caused the maximal contracting effect was used to provide vascular tone in the acetylcholine (ACh) and sodium nitroprusside (SNP) trials. After a thorough washing, the rings were allowed to stabilize at baseline tension for 20 min and then carefully removed from the chamber. The rings were then placed in tightly capped glass test tubes containing Krebs solution saturated with oxygen within a heated (37°C) shaking bath. Neutrophils (1 x 107 cells/mL), platelet-activating factor (PAF; 1 µM) (11), and platelets (1 x 108 cells/mL), when appropriate, were added to the tubes alone or together with 0.30 mM isoflurane in Krebs solution and incubated for 25 min. This millimolar concentration for isoflurane was calculated using an isoflurane 1.0 minimum alveolar concentration (MAC) of 1.4 vol % for the dog (26) and a buffer/gas partition coefficient of 0.55 at 37°C and 1 atm (27). A stable level of isoflurane during all experimental protocols was confirmed by gas chromatography. The concentrations for PAF and isoflurane in the ring studies were also used in the superoxide and adherence studies described below. The main experimental groups in the ring studies were as follows: 1) control; no neutrophils, PAF, or platelets; 2) PAF and neutrophils; 3) PAF, neutrophils, and platelets; 4) PAF, neutrophils, and isoflurane; 5) PAF, neutrophils, platelets, and isoflurane. Additional validation studies were conducted with platelets both alone and with PAF and with neutrophils alone.
After the incubation period with the inflammatory cells, the rings were assessed for endothelial function. The rings were removed from the test tubes and washed three times with Krebs solution to remove neutrophils, platelets, PAF, and isoflurane. They were then re-mounted in the chambers, and allowed to stabilize at the appropriate pre-tension for 40 min. Indomethacin (10 µM) was added to the chambers to prevent vascular responses to prostaglandins (11,32). Once a stable contraction to the predetermined U46619 dose was obtained, endothelial function was assessed from cumulative concentration-response curves to the endothelium-dependent dilator ACh (10–9 to 10–6 M). After the rings were again washed and allowed to stabilize, vascular smooth muscle function was assessed from the curves to the endothelium-independent dilator SNP (10–9 to 10–6 M). Drug concentrations are expressed as the final concentrations in the organ chamber.
Superoxide production by neutrophils in suspension was determined by measuring the superoxide dismutase (SOD) inhibitable reduction of ferricytochrome c to ferrocytochrome c (11). Neutrophils (5 x 106 cells/mL) or/and platelets (5 x 107 cells/mL) were prewarmed in a shaking incubator at 37°C with 160 µM cytochrome c and 5 µg/mL cytochalasin B in the absence (control) or presence of the test agents for 5 min and then stimulated with PAF. Half of the tubes were provided with an excess of SOD (100 µg/mL) as a control for nonspecific activity or color generation. Five min after adding PAF, cytochrome c reduction was measured spectrophotometrically by determining the optical density of the supernatant at 550 nm, using a Vmax kinetic microtiter plate reader (Molecular Devices, Palo Alto, CA). Superoxide production was calculated using an extinction coefficient of 21 mM–1cm–1 for cytochrome c. Results are expressed as nM of SOD-inhibitable O2– produced by a suspension of 5 x 106 neutrophils/mL. The experimental groups were the same as those in the endothelial dysfunction studies described above. Further validation studies were performed to determine the ability of the PAF-activated platelets alone to produce superoxide.
Neutrophil adherence to the endothelial surface of the coronary artery segments was assessed using neutrophils labeled with a vital fluorescent dye, as described previously (11). Briefly, 1 mL of 4 µM solution of PKH26 dye was added to 1 mL of a neutrophil suspension (2 x 107 cells/mL). After the sample was gently mixed, it was incubated at room temperature for 5 min; the labeling reaction was stopped by adding 2 mL plasma and incubating for 1 min. The plasma-stopped sample was diluted with 4 mL of HBSS and then centrifuged at 400g for 10 min at 4°C. The resultant cell pellet was transferred to a new tube for additional duplicate washings, and the cells were resuspended. This labeling procedure yields neutrophils possessing normal viability and function (11).
Coronary artery rings were carefully opened without damaging the endothelium and placed in the tube containing 3 mL of HBSS with Ca2+ at 37°C. Labeled neutrophils were added to each tube to achieve a final concentration of 5 x 106 neutrophils/mL. The coronary vascular segments were incubated with labeled neutrophils in the absence and presence of platelets (5 x 107 cells/mL) and isoflurane for 20 min. PAF was used as an activator of the cells. After removing the vascular segments, they were flushed gently with HBSS. Neutrophil adherence was determined by counting the number of neutrophils adhering to the endothelial surface in six separate microscopic fields under epifluorescent microscopy (490-nm excitation, 504-nm emission) (Fryer Company, Inc, Huntley, IL), and expressed per square millimeter of endothelium. The experimental groups were the same as those in the endothelial dysfunction studies described above.
The following chemicals and reagents were obtained from Sigma Chemical (St. Louis, MO): acetylcholine chloride, SNP, Ficoll-Pacque, SOD, cytochrome c, cytochalasin B, PKH26 dye, and indomethacin. PAF and HBSS without Mg2+ and Ca2+ were purchased from Avanti Polar Lipids (Alabaster, AL) and Meditech, Inc. (Salt Lake City, UT), respectively. All solutions were prepared freshly on the day of the study.
Concentration-responses for vascular relaxation were calculated as a percentage of the decrease of U46619-induced isometric force. The drug concentration required to elicit 50% of the maximum relaxation response (EC50) was calculated by linear regression analysis and expressed as the negative logarithm of the drug concentration (–log [M]). The maximal relaxation response (Rmax, %) was also determined. An Rmax value equal to 100% indicated complete reversal of U46619-induced contraction. A one-way analysis of variance in combination with the Student-Newman-Keuls test were used to evaluate treatment effects on superoxide production and neutrophil adherence (28). Within- and between-group differences for coronary vascular relaxation responses were assessed using a two-way analysis of variance for repeated measures followed by the Student-Newman-Keuls test (28). A value of P < 0.05 was considered significant throughout the study.
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Results |
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Figure 2 presents the effects of isoflurane on superoxide production by PAF-stimulated neutrophils alone and in the presence of platelets. PAF stimulation of neutrophils caused a ninefold increase in superoxide production. This response was greater in the presence of platelets. Isoflurane inhibited superoxide production by neutrophils alone and it abolished the ability of platelets to enhance this production. PAF-activated platelets alone produced a negligible amount of superoxide.
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Figure 3 demonstrates that PAF caused a pronounced increase neutrophil adherence to the coronary artery rings, which was enhanced by the presence of platelets. Isoflurane reduced adherence of the activated neutrophils alone and also abolished the platelet-induced enhancement of this effect.
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Discussion |
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There is an extensive body of evidence suggesting that interactions between neutrophils and platelets may be important in the pathophysiology of myocardial and coronary endothelial reperfusion injury (1–4). Platelet-neutrophil conjugates have been demonstrated to enhance neutrophil activation and neutrophil-induced cardiac injury (5,6), as reflected by the enhanced increases in superoxide production, vascular adherence, and the coronary endothelial dysfunction observed in the current study. Previous studies have suggested that platelets promote neutrophil activation via the release of several mediators, including thromboxane A2, platelet-derived growth factor, platelet factor 4, and serotonin (3). Although the main mechanism for platelet-neutrophil interactions is P-selectin on the surface of the platelets and PSGL-1 on the surface of the neutrophils, other mechanisms have been implicated. These include 1) fibrinogen bridging via GPIIb/IIIa on the platelet and the ß2 integrin MAC-1 (CD11b/CD18) on the neutrophil, 2) thrombospondin bridging via GPIa/IIa, GPIIb/IIIa, or GPIV on the platelet and a specific receptor on the neutrophil, and 3) platelet intercellular adhesion molecule-2 (ICAM-2) binding to neutrophil lymphocyte function-associated antigen-1 (LFA-1) (29).
Platelets can adhere to the endothelium via glycoprotein 1b on the platelet surface and von Willebrand factor in the vessel wall and this interaction can cause release of chemotactic proteins and expression of adhesion molecules in the endothelium, thus promoting neutrophil-endothelial interactions and endothelial injury (30). Although this mechanism may have played a role in our adherence and vascular relaxation studies, it could not have been involved in the superoxide studies, which did not include coronary arterial segments.
It has been demonstrated that the platelets themselves can produce superoxide but, as confirmed in our validation studies, this production is much smaller than that of the neutrophils and is likely limited to intracellular signaling (31). We also demonstrated that activated platelets in the absence of neutrophils had no effect on the relaxation responses of the coronary artery segments. These findings, when taken together, would seem to eliminate the possibility that the enhanced endothelial dysfunction caused by the platelet-neutrophil combination was attributable to an independent effect of the platelets.
Activated neutrophils produce superoxide, whose action on the coronary vasculature can result in endothelial dysfunction. Superoxide production by the neutrophil can be via adherence-independent or adherence-dependent pathways (32). Our superoxide production and vascular adherence findings suggest that the platelets enhanced both these pathways and that isoflurane abolished these effects.
The mechanisms by which isoflurane inhibited the platelet-induced enhancement of neutrophil-induced endothelial dysfunction remain to be clarified. Several potential mechanisms can be proposed:
1. Isoflurane had an inhibitory effect on the platelets, which reduced interactions between the platelets and both the neutrophils and endothelium. Previous studies are equivocal regarding a potential role for this mechanism. Some have shown that isoflurane does not affect platelet function (15), whereas others have suggested that it has an inhibitory effect at clinically relevant concentrations (19). Moreover, studies have suggested that isoflurane may have a facilitating, rather than inhibitory, effect on expression of P-selectin in activated platelets and on platelet-neutrophil binding (21). However, the use of a larger concentration for isoflurane (2 MAC), different agonists (adenosine diphosphate and thrombin receptor agonist protein TRAP-6), and cells contained in whole blood samples from a different species (humans) make it difficult to extrapolate these latter findings to the present study. Although isoflurane has been shown to reduce thrombin-induced platelet adherence to the coronary vascular endothelium in isolated guinea pig hearts (19), it has also been shown to increase expression of glycoprotein 1b in adenosine diphosphate-activated platelets in vitro (23). This suggests that the former finding may reflect an inhibitory effect on the endothelium rather than on the platelets.
2. Isoflurane had an inhibitory effect on the neutrophils that rendered them unresponsive to the stimulatory action of the platelets. PAF binds to a specific receptor on the neutrophil membrane, which is the first event in the signal transduction sequence. Ultimately, the production of superoxide by the neutrophil results from activation and assembly of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, which is a transmembrane electron transport chain that reduces oxygen to superoxide. The isoflurane-induced inhibition of superoxide production by neutrophils alone in the current study could be attributable to a direct inhibitory effect on NADPH oxidase or to an inhibitory effect at some site in the signal transduction pathway regulating NADPH oxidase, e.g., the PAF receptor on the neutrophil membrane or the GTP-binding proteins (G proteins) that are involved in transduction of agonist signals. This inhibitory action of isoflurane on NADPH oxidase may prevent modulation from upstream stimulatory mediators, including those released by the platelets.
3. Isoflurane had a protective effect on the coronary endothelium that prevented the dysfunction caused by activated neutrophils both alone and in the presence of platelets. Free radicals released from neutrophils, e.g., superoxide, provide a stimulus for up-regulation of endothelial adhesion molecules (33). The main endothelial adhesion molecules are P-selectin, which mediates the initial "rolling" and slowing of neutrophils along the endothelial surface, and ICAM-1, which mediates the later firm adherence of neutrophils to the endothelial cell surface (11). In the PAF-stimulated in vitro system used in the current study, adherence is via ICAM-1 (32). We have demonstrated in an isolated rat heart preparation (in the absence of platelets) that selective exposure of the coronary vasculature to isoflurane can reduce adherence of activated neutrophils (10). Although not confirmed by direct measurements, this reduced adherence would likely have resulted in better preserved endothelial function. Exposure to isoflurane may protect the coronary endothelium from the deleterious effects of activated neutrophils both in the absence and presence of platelets.
Our findings confirm our previous work indicating that isoflurane has a profound inhibitory effect on neutrophil activation and functions (11). In this regard, it is noteworthy that isoflurane was capable of attenuating, but not abolishing, the increase in superoxide production by the activated neutrophils, whereas it could completely abolish the neutrophil-induced endothelial dysfunction and the associated increases in neutrophil adherence. This apparent quantitative divergence can be reconciled if the ability of activated neutrophils to interact and cause dysfunction within the endothelium was a threshold phenomenon and isoflurane was able to reduce neutrophil activation below the critical level.
PAF is a highly active phospholipid that has been implicated in inflammatory responses and cardiac reperfusion injury in vivo (34). Exogenous PAF was used as an agonist in the present study because of its well-established ability to activate the three cell types investigated: neutrophils, platelets, and endothelial cells. We followed previous studies and treated the coronary vascular rings with indomethacin to inhibit prostaglandin synthesis (11,32). Although indomethacin has been demonstrated to increase the sensitivity of canine coronary arteries to the constrictor effects of U46619 (35), there is no reason to expect that this would have influenced our results or conclusions.
A blunted response to the endothelium-dependent vasorelaxing drug ACh could reflect impaired endothelial or vascular smooth function. SNP, a nitric oxide donor, is an endothelium-independent vasorelaxing drug that was used to differentiate between these possibilities. The inability of neutrophils alone, or combined with platelets, to attenuate the responses to SNP implied well preserved vascular smooth function and suggested endothelial dysfunction as an explanation for the blunted responses to ACh.
In a previous investigation (11), we performed a control study that indicated that treatment with isoflurane alone did not alter the ACh vasorelaxation curve. This finding eliminates the possibility that isoflurane’s beneficial effect on ACh-induced coronary relaxation in the studies involving activated neutrophils and platelets was caused by an enhancement of baseline endothelial function and was not related to protection against neutrophil- and/or platelet-induced endothelial damage.
In conclusion, the present study demonstrated that isoflurane can inhibit the ability of platelets to enhance neutrophil-induced vascular endothelial dysfunction in the coronary circulation. Our findings provide additional support for the notion that the cardioprotective effects of isoflurane involve inhibitory actions on inflammatory cells and their interactions, as well as KATP channel-dependent signaling pathways within myocytes (8).
The authors thank Derrick L. Harris, BS, for technical assistance.
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Footnotes |
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Presented in part at the annual meeting of the American Society of Anesthesiologists, Orlando, Florida, October 12–16, 2002.
Accepted for publication June 10, 2005.
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References |
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B and AP-1 and upregulation of ICAM-1 in reperfused canine heart. Am J Physiol 2002;282:H1778–86.
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