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*Department of Anaesthesiology and Intensive Care and
Institute of Clinical Chemistry and Laboratory Medicine, University Hospital Münster, Münster, Germany
Address correspondence and reprint requests to Maike A. Große Hartlage, MD, Universitätsklinikum Münster, Klinik und Poliklinik für Anästhesiologie und Operative Intensivmedizin, Albert-Schweitzer-Straße 33, 48149 Münster, Germany. Address e-mail to grosse.hartlage{at}anit.uni-muenster.de
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IMPLICATIONS: Inhalational administration of 75 vol% xenon provides cardiovascular stability and improves recovery from myocardial stunning in chronically instrumented dogs under fentanyl/midazolam anesthesia. This suggests that xenon may be a safe anesthetic for patients at high risk of perioperative myocardial ischemia.
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Introduction |
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Methods |
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Ten healthy, heartworm-free foxhounds of either sex, weighing 23–25 kg, were selected for the studies. After overnight fasting, but with free access to water, the dogs received IM premedication with piritramide 1 mg/kg and ketamine 5 mg/kg. Anesthesia was induced with IV propofol 7 mg/kg and fentanyl 20 µg/kg. A cuffed endotracheal tube was inserted, and mechanical ventilation was adjusted to maintain arterial blood gas tension values within the physiologic range. Anesthesia was maintained with isoflurane in a mixture of oxygen and air, supplemented with intermittent application of fentanyl 10 µg/kg. To prevent hypovolemia, lactated Ringer’s solution was infused at a rate of 12 mL · kg–1 · h–1. Body temperature was maintained by the use of a heating pad. Perioperative antibiotic prophylaxis was achieved with cefamandole 30 mg/kg for 3 days.
A left thoracotomy was performed in the fifth intercostal space under strictly aseptic conditions. The left lung was retracted, and the heart was exposed. The aorta and the left atrium were cannulated with 18-gauge Tygon catheters for measurement of pressures, injection of microspheres, and withdrawal of blood. Left ventricular pressure (LVP) and the rate of rise of LVP were determined with a pressure microtransducer (Janssen Pharmaceutica, Beerse, Belgium) inserted into the left ventricle (LV) through an apical stab incision. Pulsed Doppler blood-flow velocity (BFV) probes (20 MHz; Baylor College of Medicine, Houston, TX) were placed around the left anterior descending coronary artery (LAD). Although this method does not measure absolute blood flow, there is an excellent correlation between Doppler frequency shift and absolute blood flow. Proximal to the Doppler flowprobe, a pneumatic occluder was positioned around the LAD to induce reversible ischemic episodes in the LAD-perfused myocardium. To assess the regional myocardial wall thickening, 10-MHz pulsed Doppler crystals (Baylor College of Medicine) were sutured precisely perpendicular and flat to the epicardium of the LAD-perfused area. This atraumatic technique is based on principles of pulsed echo and Doppler ultrasound and has been described in detail previously (11,12). A validation for myocardial ischemia and reperfusion studies has been performed (11). At the end of the surgical instrumentation, the pericardial edges were approximated, and the thorax was closed in layers. All leads were tunneled subcutaneously and exteriorized between the scapulae. Postoperative analgesia was provided with IM-administered piritramide.
After instrumentation, the animals were trained daily to accustom them to the experimental environment. Experiments were performed after a postoperative recovery time of at least 12 days.
Aortic and left atrial pressures (LAP) were measured with disposable pressure transducers. Pressures, blood-flow velocity, and wall-thickening signals were processed by using a six-channel pulsed Doppler system (Baylor College of Medicine).
The wall-thickening fraction (WTF) for a myocardial layer is the ratio between systolic thickening and the thickness of that layer (11). Systolic thickening is defined as the maximal excursion of the myocardial wall recorded during systole. Using the pulsed Doppler method, WTF is calculated as
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When measuring transmural thickening, the Doppler technique requires the range-gated sample volume to be positioned at a depth in the ventricular wall that is close to that of the endocardium at end-diastole. The LVP signal was electronically differentiated (Gould Inc., Cleveland, OH) to obtain the maximum rate of LVP increase (LV dP · dtmax–1) and decrease (LV dP · dtmin–1). All signals were recorded digitally.
The transmural distribution of the regional myocardial blood flow (RMBF) was assessed by the use of fluorescent microspheres (NuFLOWTM microspheres; Interactive Medical Technologies, Ltd., Irvine, CA), a technique that has been validated previously (13). The RMBF was measured three times in each experiment: 1) baseline under anesthesia; 2) during ischemia, 3 min after LAD occlusion; and 3) after the third minute of reperfusion. Before injection, the microspheres were agitated with a vortex mixer and in an ultrasonic bath to prevent aggregation. For each measurement, 9 x 106 microspheres of a certain color and with a diameter of 15 µm were applied. They were injected into the left atrial appendage over 1 min to guarantee that they were well mixed in the coronary blood supply. The reference blood sample that is needed to calculate the RMBF was drawn from the aortic catheter at a constant rate of 6.6 mL/min by use of a precalibrated withdrawal pump (Harvard Apparatus, South Natick, MA). Blood withdrawal started 10 s before microsphere injection and continued for 2 min after injection. The application of this quantity of microspheres caused no changes in coronary blood flow, thus implying unaltered coronary hemodynamics, and it also did not influence HR, mean arterial blood pressure (MAP), LV dP · dtmax–1, or LV dP · dtmin–1.
When the regional myocardial function had completely recovered after the second ischemic episode, animals were killed during general anesthesia by infusion of potassium chloride, and the heart was excised. Transmural tissue samples of 1–2 g were obtained from the LAD-perfused area in the immediate vicinity of the implanted wall-thickening probes and further dissected into the endocardial and epicardial layer. Samples from the area that was normally perfused by the circumflex branch of the left coronary artery (LCX) served as controls. Extraction and measurement of microspheres in the samples were performed in an independent laboratory by flow cytometer analysis (Interactive Medical Technologies, Ltd.). For each heart, the induced ischemia was considered to be severe enough only if the tissue samples from the LAD-perfused myocardium had an RMBF 
25% of the corresponding nonischemic RMBF under baseline conditions. The endocardial/epicardial RMBF ratio was calculated for the LAD- and the LCX-perfused myocardium.
Arterial blood samples for measurement of plasma catecholamines were obtained at the following time points: under preanesthetic baseline conditions; during steady-state anesthesia 30 min after induction; under ischemia; and 1, 5, 15, 30, 60, and 180 min after reperfusion. Five milliliters of blood was collected into prechilled polystyrene syringes that contained reduced glutathione and EGTA (KABE Labortechnik, Nümbrecht-Elsenroth, Germany). The samples were centrifuged at 3000 rpm over 20 min at 4°C, and the plasma was separated immediately and stored at –70°C until analysis. EPI and norepinephrine (NE) were assayed by using the high-performance liquid chromatography technique. Samples of 1 mL of plasma were prepared with commercially available high-performance liquid chromatography assay kits consisting of an extraction buffer, 3,4-dihydroxybenzylamine (used as an internal standard to monitor recovery from the extraction step), a wash buffer, and an elution buffer, all provided by Chromsystems (Munich, Germany). Catecholamines were selectively isolated from the gained eluate by adsorption on activated alumina columns according to the method described by Maycock and Frayn (14). Forty microliters of the eluate was injected on an isocratic Kontron 422 liquid chromatograph (Kontron Instruments, Neufahrn, Germany) interfaced with a Model 41000 electrochemical detector (Chromsystems). The potential of the glassy carbon working electrode of the detector was set at 0.5 V against an Ag/AgCl reference electrode. The catecholamines were separated on reversed-phase columns (length, 100 mm; interior diameter, 4 mm) at a constant flow rate of 1 mL/min at room temperature.
The lower detection limit, as defined by 95% of the upper plateau of the standard curve, was 10 pg/mL per tube for EPI and NE. The intraassay and interassay coefficients of variation were 5.4% and 10.6%, respectively, for EPI and 5.8% and 9.4%, respectively, for NE.
All animals were subjected to two experimental conditions, control and intervention, in a randomized crossover design on separate days. Using a computer-generated random-number table, the animals were assigned to 1 of 2 groups (A and B), each consisting of 5 animals. The five dogs of Group A were first subjected to the control experiment and then to the intervention, whereas in Group B, the intervention was performed first and then the control experiment. The number of ischemic episodes was restricted to two in each animal, because multiple stunning maneuvers may induce development of coronary collaterals and thus prevent the induction of postischemic dysfunction. The interindividual variability in canine coronary anatomy was taken into consideration, because the control experiments and the intervention were performed in the same animals and not in separate experimental groups. The minimum interval between the 2 experiments was 6 days to avoid a possible preconditioning (PC) effect of the first ischemic episode (15).
The duration of each ischemic episode was chosen to be 10 min to cause myocardial stunning of relevant severity (16,17) and at the same time to prevent the induction of myocardial infarction (18). Each dog had one 10-min LAD ischemia during anesthesia with midazolam and fentanyl (control) and a second 10-min ischemic episode under the same basal anesthesia but with concomitant inhalational application of 75 ± 1 vol% xenon of medical quality (Messer Griesheim GmbH, Krefeld, Germany), corresponding to 0.66 minimal alveolar anesthetic concentration (MAC) in dogs (19). Because of nitrogen accumulation in the closed-circuit ventilator, an inspiratory xenon concentration of 79 vol% was not achieved.
Basal anesthesia was induced with initial IV loading doses of fentanyl (20 µg/kg) and midazolam (0.4 mg/kg) and was maintained with continuous IV application of fentanyl 25 µg · kg–1 · h–1 and midazolam 0.6 mg · kg–1 · h–1. The adequacy of this anesthesia regimen was demonstrated in previous experimental studies (20). After tracheal intubation, mechanical ventilation was performed with a special closed-circuit ventilator, a modified PhysioFlex® (Draeger, Lübeck, Germany), in both experiments. The continuous measurement of the xenon concentration by the PhysioFlex® was based on a thermal conductivity method (xenon sensor Type 6S22 WPI XE IT; Sensor Devices GmbH, Dortmund, Germany) (21). The ventilator has been used successfully in other xenon investigative studies (22). Animals were ventilated with a minute volume adjusted to achieve a PaCO2 of 34–37 mm Hg. A fraction of inspired oxygen of 21 vol% was chosen, guaranteeing a sufficient oxygenation, with a mean PaO2 of 96 mm Hg controlled by blood gas tension analyses. The concomitant administration of xenon started immediately after tracheal intubation, and in both experiments, LAD ischemia was induced under steady-state anesthesia 35 min later. Anesthesia was discontinued in both groups after 20 min of reperfusion. On average, animals began to breathe spontaneously after 30 min of reperfusion and could be tracheally extubated 15 min later. During this period, arterial blood analyses were performed, and no changes in arterial oxygen partial pressure were observed.
Data acquisition was performed at the following predetermined time points: in the awake state (preanesthetic baseline); under steady-state anesthesia 30 min after tracheal intubation (baseline under anesthesia); during ischemia; and 1, 5, 10, 15, 20, 30, 45, 60, and 90 min and 2, 3, 6, 12, 24, and 48 h after the beginning of reperfusion. At each of these time points, hemodynamic variables and wall-thickening data were recorded over 30 s. Data presented are mean values of these recordings and not single values at specified time points.
Since we regarded only a very large positive effect of xenon on recovery from myocardial stunning as important, we purposefully chose a very small sample size. A post hoc power analysis was performed (power = 88%; 
= 0.05; effect size 
= 0.7) and required a minimum of 10 dogs.
To analyze treatment effects by comparison of different points of measurement, statistical analysis was performed by using one-way analysis of variance for repeated measurements and Student’s t-test for dependent samples. P values of <0.05 were considered significant. Data are presented as mean ± SD.
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Results |
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There were no significant changes of the transmural systolic WTF in the LAD-perfused myocardium after the induction of anesthesia in either group (Fig. 1). Regional ischemia led to a significant reduction of WTF to negative values (wall thinning) in the control experiments (–35% ± 23%) and under xenon anesthesia (–33% ± 18%). In the group receiving xenon, reperfusion resulted in an immediate complete recovery of WTF. Without xenon, WTF returned after reperfusion on the average only to 46% of the baseline level, and preischemic WTF values were reached after 48 h. Dogs receiving xenon showed a significantly better recovery of WTF up to 12 h after ischemia. Discontinuation of anesthesia had no influence on regional myocardial WTF in either experiment. The positions of the wall-thickening probes in the LAD-perfused area were confirmed in the postmortem inspection of the heart to be located on the tissue samples included for analysis of LAD transmyocardial blood-flow distribution.
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Both anesthetic regimens resulted in a significant decrease in MAP and HR compared with baseline values, whereas LAP remained unchanged (Table 3). In both groups, the induction of ischemia did not change MAP and HR in comparison to the preischemic state, whereas LAP increased significantly. After reperfusion, MAP and HR remained significantly reduced compared with baseline until emergence from anesthesia. The LAP, however, showed no deviation from baseline levels during the entire reperfusion period (Table 4).
Preanesthetic baseline levels of NE were 187 ± 27 pg/mL in the xenon group and 215 ± 31 pg/mL in the control group (Fig. 2). After the induction of anesthesia, NE levels increased significantly in the control experiment and the intervention. After discontinuation of anesthesia, NE levels decreased but were still significantly increased during emergence from anesthesia and 1 h after reperfusion. NE values in the xenon group did not return to baseline 3 h after reperfusion. Differences between groups were not observed. Preanesthetic EPI levels were 36 ± 10 pg/mL in the xenon group and 45 ± 12 pg/mL in the control group (Fig. 3). EPI values were not influenced by the induction of anesthesia and ischemia in both experiments. During emergence from anesthesia, EPI levels increased significantly and peaked 30 min after reperfusion with 314 ± 37 pg/mL in the xenon group and 436 ± 56 pg/mL in the control group. Thereafter, EPI values decreased but did not return to baseline levels 3 h after reperfusion in either group. A comparison between groups showed that 30 and 60 min after reperfusion, EPI levels were lower after xenon anesthesia.
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Discussion |
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In studies on healthy humans during clinical anesthesia (4,5), as well as in experimental studies (3,9), xenon has been reported to be associated with cardiovascular stability. Xenon did not impair the responsiveness of isolated ventricular muscle bundles to positive inotropic and chronotropic stimulation (23). In ASA class I patients undergoing abdominal surgery, Luttropp et al. (5), using transesophageal echocardiography, demonstrated that LV fractional area change was unaffected by 65 vol% xenon. In isoflurane-anesthetized healthy dogs, xenon did not affect intrinsic myocardial contractility (9). In accordance with our results, LV dP · dtmax–1 remained unchanged. The reported diastolic dysfunction, with a decrease in the magnitude of LV dP · dtmin–1 and an increase in 
, which occurred dose-dependently under 65 vol% xenon (9), was not seen in this investigation. In acutely instrumented dogs under midazolam/piritramide anesthesia, intracoronary administration of xenon produced a small direct negative inotropic effect (7). These apparent discrepancies with our results are likely attributable to methodological differences, including those related to the experimental model (acute versus chronic) and different background anesthetics.
The decrease in LV dP · dtmax–1 and LV dP · dtmin–1 during anesthesia and the early reperfusion period can be explained by the global negative inotropic effect of the combination of fentanyl and midazolam. LV dP · dtmax–1 and LV dP · dtmin–1 remained unchanged after the induction of ischemia and reperfusion in both groups because no global contractile dysfunction was induced. The WTF data, however, reflect severe regional myocardial dysfunction.
Several studies investigating xenon have reported that the HR has a tendency to decrease (5,6). Dogs receiving xenon in our study also showed a tendency for HR reduction. Conversely, xenon did not prevent the reactive, albeit insignificant, increase in HR during coronary occlusion. It must be considered that the combination of fentanyl and midazolam was primarily responsible for a constant slow HR and decreased MAP. The R-R intervals in the electrocardiograph were evaluated, but xenon did not induce detectable arrhythmias.
Detailed information about the cardiovascular effects of xenon under pathophysiological conditions such as myocardial ischemia are scarce. Inhalation of 70 vol% xenon during early reperfusion reduced infarct size after regional ischemia in -chloralose-anesthetized, acutely instrumented rabbits (8). In the cited investigation, however, xenon was administered only during the first 15 minutes of reperfusion, whereas in our study ischemia and reperfusion both occurred during xenon anesthesia and thus resembled a clinical scenario of intraoperative myocardial ischemia.
The most important finding of this study is that xenon improves functional recovery from regional myocardial stunning. Severe ischemia was confirmed by a reduction of subendocardial perfusion to <20% of baseline values and by a significant reduction of WTF to negative values. Thus, the cardioprotective effect of xenon occurred without preventing the depression of systolic wall function that occurs during ischemia.
Our data do not allow an analysis of the underlying mechanism; however, several possibilities warrant consideration. One theory regarding the pathogenesis of myocardial stunning postulates that the contractile dysfunction is a result of a disturbance of cellular calcium homeostasis, with intracellular calcium overload and decreased responsiveness of the contractile proteins to calcium (24). In electrophysiologic studies on cardiomyocytes, however, it was reported that xenon does not alter the major cardiac cation currents (25,26). Xenon 80 vol% did not exhibit any measurable effect on Na+, L-type Ca2+, or inward-rectifier K+ channels in isolated ventricular myocytes of the guinea pig (25). In human atrial cardiomyocytes, xenon did not affect voltage-gated Ca2+ currents and only slightly inhibited transient K+ outward currents (26).
It has been suggested that volatile anesthetics mediate ischemic PC by opening the myocardial adenosine triphosphate (ATP)-sensitive potassium channels (KATP) (27). No data have been published regarding the effects of xenon on myocardial KATP. In addition, several investigations confirmed that not the early, but only the late phase of PC protects against stunning. Furthermore, adenosine and KATP play solely an obligatory role in ischemia-induced late PC against infarction, but not against stunning (28). On this scientific background, the improved recovery from stunning after inhalational application of xenon cannot be explained by a PC effect mediated by adenosine and KATP.
Catecholamine plasma levels were measured to further characterize and interpret the mechanisms underlying the effect of xenon on the recovery from myocardial stunning. The very few investigations that are directed at the neurohumoral effects of xenon anesthesia point to a central attenuation of the neurohumoral response to surgical stress (3,6). Marx et al. (3) investigated dose-dependent (30–70 vol%) influences of xenon anesthesia in mechanically-ventilated barbiturate-anesthetized pigs subjected to standard surgical stress. Whereas dopamine and NE concentrations remained within normal limits, EPI concentrations during xenon anesthesia were significantly reduced not only at inspiratory concentrations of 1 MAC, but also at subanesthetic concentrations of 30 and 50 vol%. These reports are not directly comparable with our experiments because we used chronically instrumented dogs and caused no surgical stress. Nevertheless, in our model of myocardial ischemia and reperfusion injury, xenon also displayed favorable neurohumoral properties. In the xenon group, plasma EPI levels were significantly lower than control values during emergence from anesthesia and in the early postanesthetic phase. Studies from our group have shown that a decrease in sympathetic activity improves recovery from myocardial stunning (17). However, it is very unlikely that the presented catecholamine data are an explanation for the improved recovery from myocardial stunning after xenon administration. During ischemia and in the early reperfusion period, EPI and NE values did not correspond to the time course of the WTF values. Neither the increase of the EPI plasma levels during emergence from anesthesia, nor the significantly different EPI levels in the xenon and control groups are reflected in the WTF data. In this context, it is notable that the increased EPI plasma levels during emergence from anesthesia are not reflected in systemic hemodynamic values. We postulate that the global EPI effects were blunted by the preceding opioid/benzodiazepine anesthesia. In both experiments, NE plasma values increased significantly after the induction of anesthesia and decreased after its discontinuation. Because fentanyl/midazolam anesthesia causes vasodilation, the increased release of NE during anesthesia can most likely be explained as a counterregulatory reaction aimed at MAP stabilization.
An important effect of inhaled anesthetics is the decrease of coronary vascular resistance (20). However, coronary artery blood flow was unaffected by xenon in isolated guinea pig hearts (25). Very little is known about the effects of xenon on myocardial tissue perfusion (7,29). In an investigation by Preckel et al. (7), regional intracoronary administration of 70 vol% xenon in acutely instrumented dogs did not affect the transmural blood-flow distribution pattern. Our present data are in accordance with these previous findings, showing that xenon caused no significant decline of the RMBF in the LAD- and RCX-perfused myocardium. In addition, xenon did not affect the endocardial/epicardial blood-flow ratio in normally perfused myocardium or during ischemia. The observed improved recovery from myocardial stunning cannot be attributed to effects on myocardial perfusion.
Some aspects of the experimental model used in this study require consideration for interpretation of our data. Because xenon was administered systemically, it is not possible to distinguish the direct effects of xenon on myocardial function from indirect effects due to changes in global hemodynamics. However, our data demonstrate, in good correlation with previous investigations, that xenon does not alter systemic hemodynamics.
Under both experimental conditions, ventilation was performed with a closed-circuit ventilator. The end-expiratory xenon concentrations were displayed, whereas measurements of the xenon concentration in the blood reaching the LAD were not available. However, when similar measurements were obtained in other studies, the anesthetic blood concentrations were proportional to the percentage of inhaled anesthetic (30).
Another important consideration in the interpretation of our data is that we investigated the effects of an inhaled anesthetic under the influence of an opioid and a benzodiazepine. A legitimate concern, therefore, is that the results may have been influenced by the different pharmacodynamic and pharmacokinetic properties of these IV drugs. However, the MAC of xenon varies among species, and Eger et al. (19) reported a MAC of 119 vol% in dogs. Thus, general anesthesia with xenon has to be supplemented with other anesthetics in this species. Several studies in dogs have successfully used fentanyl/midazolam anesthesia to assess the cardiovascular and coronary effects of volatile anesthetics (20). This anesthetic regimen is associated with only minimal effects on coronary blood flow, myocardial contractility, and hemodynamic state (31,32). In addition, Kato and Foex (33) showed in Langendorff rat hearts that fentanyl reduced infarct size, but did not protect against myocardial stunning.
The possibility that the between-group differences seen in WTF may be explained by a greater anesthetic depth in the xenon group is very unlikely. During anesthesia, there were no differences regarding any hemodynamic variable or catecholamine plasma levels between groups. Vegetative reactions were the same under both anesthetic regimens, and arousal from anesthesia followed the same time course after the control experiment and the intervention.
In conclusion, our study demonstrated under controlled hemodynamic conditions that the inhalational administration of 75 vol% xenon improves recovery from myocardial stunning in chronically instrumented dogs under fentanyl/midazolam anesthesia. The characteristic cardiovascular stability provided by xenon anesthesia and its protective effects against postischemic contractile dysfunction suggest that xenon could be a safe anesthetic, especially for patients at high risk of perioperative myocardial ischemia.
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Acknowledgments |
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  J.-H. Baumert, M. Hein, K. E. Hecker, S. Satlow, P. Neef, and R. Rossaint Xenon or propofol anaesthesia for patients at cardiovascular risk in non-cardiac surgery Br. J. Anaesth., May 1, 2008; 100(5): 605 - 611. [Abstract] [Full Text] [PDF]
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