Myocardial infarct size is a strong, independent predictor of heart failure as well as early and late survival after acute myocardial infarction.1,2 Therefore, the development of novel strategies capable of maximizing the salvage of myocardium during reperfusion therapy could be of great epidemiologic and economic significance. Such strategies might also be of value following cardiac surgery or successful resuscitation from cardiac arrest.
Postischaemic application of xenon 70% (postconditioning stimulus) has been shown to reduce myocardial infarct size in rabbits.3 The combination of subanaesthetic concentration (20%) of xenon and mild hypothermia of 35°C significantly reduced tissue damage when administered after ischaemic–hypoxic brain injury in neonatal rats.4 This neuroprotective effect was evident not only when hypothermia and xenon were administered simultaneously, but also when administered asynchronously.5 In both settings, neither intervention was efficacious when administered separately.
As emergency treatment immediately after an acute ischaemia–reperfusion event, that is, as an adjunct to reperfusion therapy following myocardial infarction or successful resuscitation from cardiac arrest, subanaesthetic tissue concentrations of xenon can be established rapidly by inhalation. In contrast, even mild myocardial hypothermia is difficult to achieve without significant time delay relative to the onset of reperfusion. It is likely that hypothermia, if applied as a sole intervention, is cardioprotective only if it is already established during the first minutes of reperfusion.6 We speculated that an organ protective synergism between xenon and hypothermia, as has been demonstrated in the brain, could also work for the benefit of reperfused myocardium. If that was to be the case, then xenon inhalation could be of therapeutic value by ‘bridging’ the delay from the onset of reperfusion until hypothermia is established.
The present study was designed to investigate the hypotheses that the combination of a subanaesthetic concentration of xenon 20% and mild hypothermia of 34°C, applied at the onset of reperfusion, reduces myocardial infarct size; the two component interventions produce an infarct-reducing effect if applied asynchronously, that is, xenon inhalation followed by hypothermia.
The study was performed in accordance with the regulations and guidelines of the Animal Ethics Committee of the University of Amsterdam, The Netherlands, and was in line with European Union directives on the care and use of experimental animals. We obtained institutional review board approval from the Animal Ethics Committee of the University of Amsterdam to perform this study. Animals had free access to food and water at all times before the start of the experiments.
Xenon was kindly provided by Linde AG (Linde Gas Therapeutics, Eindhoven, The Netherlands). All chemicals and reagents were purchased from Sigma (Taufkirchen, Germany).
Surgical preparation was performed as described previously.6 In brief, male Wistar rats (275–350 g) were anaesthetized by intraperitoneal injection of S-ketamine (150 mg kg−1) and diazepam (1.5 mg kg−1). S-ketamine does not interfere with preconditioning in animals in vivo.7 After tracheal intubation, the lungs were ventilated with oxygen-enriched air and a positive end-expiratory pressure of 2–3 cmH2O. PCO2 and PO2 were kept within physiological limits by adjusting ventilation parameters. Body temperature was maintained at 37.3(0.3)°C by using a heating pad. The right jugular vein was cannulated for saline and drug infusion, and the left carotid artery was cannulated for measurement of aortic pressure. Anaesthesia was maintained by continuous α-chloralose infusion. A lateral left-sided thoracotomy followed by pericardiotomy was performed and a ligature (5–0 Prolene) was passed around the left anterior coronary artery. To exclude massive myocardial infarction the ligature was placed below the first major branch of the left anterior coronary artery. All animals were left untreated for 20 min before the start of the respective experimental protocol. Arterial pressure readings were digitalized using an analogue to digital converter (PowerLab/8SP, ADInstruments Pty Ltd, Castle Hill, Australia) at a sampling rate of 500 Hz and were continuously recorded on a personal computer using Chart for Windows v5.0 (ADInstruments).
Study groups and experimental protocol
The group allocation of rats was randomized (Fig. 1). All rats underwent 25 min of coronary artery occlusion followed by 2 h of reperfusion. This period of myocardial ischaemia has been shown previously to induce myocardial necrosis in our particular model.6,8
Controls receiving 80% oxygen during reperfusion (Con80) (n = 8)
After surgical preparation, rats received no further treatment. Three minutes prior to the onset of reperfusion, the FIO2 was increased to 0.8 and maintained at this level until the end of the experiment. This FIO2 was chosen to mimic emergency treatment, when clinicians would be likely to administer as high an oxygen concentration as 20% xenon admixture would allow.
Xenon and hypothermia group (Xe20 + Hypo34) (n = 9)
These rats received xenon 20% along with hypothermia of 34°C. Administration of xenon 20% (equivalent to 0.12 minimal alveolar concentration in rats) and oxygen 80% was commenced 3 min prior to, and discontinued 30 min after, the onset of reperfusion. From previous studies we know that this time period is long enough to induce cardioprotection by inhalational anaesthetics.3,9 Following cessation of xenon inhalation, the FIO2 of 80% was maintained until the end of the experiment. Active cooling was commenced 5 min prior to, and hypothermia maintained for 1 h after, the onset of reperfusion.
Hypothermia 34°C (Hypo34) (n = 8), xenon 20% (Xe20) (n = 8)
These rats received xenon 20% alone or hypothermia of 34°C alone. From previous studies it is known that sub-anaesthetic concentrations of xenon or mild hypothermia alone did not induce organ protection.4,5
Asynchronous xenon and hypothermia group (Xe20→Hypo34) (n = 7)
These rats received xenon 20% as described above. Active cooling was commenced 5 min prior to, and hypothermia maintained for 1 h after, cessation of xenon inhalation.
Instruments of temperature regulation and measurement
At induction of hypothermia, rats were placed on a bi-layer mat fashioned from polystyrene and sheet metal. This arrangement was shown in pilot experiments to facilitate rapid surface cooling using ice-water-filled packs. Subsequent thermoregulation of the animals was achieved by using the packs in conjunction with heating lamps. At the end of the hypothermic interval rats were returned onto the heating pads and no further active thermoregulatory measures were undertaken. Thoracotomies were covered with gauze and aluminium foil.
Temperature was measured via the oesophageal (GTH 1160, Digital Thermometer, Greisinger Electronic, Germany) and rectal (ama-digit ad 15th digital thermometer, Germany) routes. Oesophageal temperature measurements, as opposed to those obtained rectally, were found to accurately reflect intrathoracic temperature at all times during pilot experiments. Oesophageal measurements were therefore considered to represent an acceptable approximation of myocardial temperature.
Infarct size measurement
Infarct size was determined using the triphenyltetrazoliumchloride staining as previously described. After 120 min of reperfusion, hearts were excised during deep anaesthesia and mounted on a modified Langendorff apparatus for perfusion with normal saline via the aortic root at a perfusion pressure of 80 cmH2O in order to wash out intravascular blood. After 2 min of perfusion, the coronary artery was re-occluded and the remainder of the myocardium was perfused through the aortic root with 0.5% Evans blue in normal saline for 10 min. Intravascular Evans blue was then washed out by perfusion for 10 min with normal saline. This treatment identified the area at risk as unstained. Hearts were then frozen and cut into transverse slices of 1 mm thickness. The slices were incubated in 0.75% triphenyltetrazolium chloride solution for 10 min at 37°C, and fixed in 4% formalin solution for 16 h at room temperature. The area of risk and the infarcted area were assessed by planimetry using SigmaScan Pro 5 computer software (SPSS Science Software, Chicago, IL).
Data are expressed as mean (SD). Heart rate [HR (beats min−1)], mean and systolic aortic blood pressure [AoPmean, AoPsyst (mmHg)] and temperature (°C) were measured during baseline, coronary artery occlusion, and the reperfusion period. Rate pressure product (RPP) was calculated as HR × AoPsyst. Comparisons of haemodynamics between groups or between time points within a group were performed using two-way analysis of variance (ANOVA; SPSS Science Software, version 12.0.1). If an overall significance was found, comparisons between groups were performed for each time point using one-way analysis of variance followed by Tukey's post-hoc test. Time effects within each group were analysed by repeated-measures ANOVA followed by the Dunnett post-hoc test with the baseline value as the reference time point.
Infarct sizes were analysed by Student's t-test followed by Bonferroni correction for multiple testing. Intervention groups (Xe20 + Hypo34 and Xe20→Hypo34) were compared with control (Con80).
Changes within and between groups were considered statistically significant if P < 0.05.
There were no significant differences in body weight, size of the area at risk, or dry weight of the hearts between the groups (Table 1).
The combination of xenon 20% and hypothermia 34°C significantly reduced infarct size [Xe20 + Hypo34: 55(22)%] compared with control [Con80: 76(12)%, P = 0.03] (Fig. 2). Xenon and hypothermia in succession produced no infarct size reduction [Xe20→Hypo34: 65(13)%, P = ns vs. controls].
Efficacy of temperature regulation
The oesophageal temperatures were 37.5°C in all groups during baseline and ischaemia (Fig. 3). Induction of hypothermia led to a rapid decline in body temperature, resulting in oesophageal temperatures of 34.8(0.3)°C at the onset of reperfusion in groups (Hypo34) and (Xe20 + Hypo34), respectively. The target temperature of 34°C was achieved 1.5(0.6) min later. The temperature curves passed their respective nadir at 32.1°C and levelled out then towards target temperature. In the group receiving first xenon and later hypothermia, the temperature decline was similar to the aforementioned changes, but 30 min later during reperfusion.
In the groups subjected to hypothermia, the haemodynamic response to cooling was near identical with respect to the depth of hypothermia (Table 2). During exposure to 34°C of hypothermia, HRs were reduced and the associated RPP at the same time points also decreased. By the end of the experiments, RPP recovered to values equal to the nonhypothermic groups.
In the present study we demonstrated that xenon at the subanaesthetic concentration of 20% and mild hypothermia of 34°C do not constitute effective postconditioning stimuli if administered as sole interventions. However, if administered in combination, they act synergistically to protect the myocardium against reperfusion injury. Asynchronous administration of the interventions – that is early xenon followed by delayed hypothermia – was not protective in our model.
For the purposes of myocardial postconditioning, beneficial effects of hypothermia seem to be lost if cooling is delayed by as little as 15 min beyond the onset of reperfusion.10,11 In the emergency situation, for example following successful resuscitation from cardiac arrest or during reperfusion therapy following acute myocardial infarction, such a delay is almost inevitable, since the event is rarely anticipated and the institution of even mild hypothermia in humans takes considerable time. Xenon, in high concentrations, has been shown to offer cardioprotective effects in different animal models.3,8,12,13 Ma et al.4 have demonstrated that sub-anaesthetic concentrations of xenon as well as mild hypothermia, when applied as sole interventions, do not protect the brains of neonatal rats against reperfusion injury. Both interventions in combination, however, act synergistically to create an effective postconditioning stimulus.4 We speculated that inhalation of very low concentrations of xenon – not efficacious by itself – may be capable of producing true therapeutic synergism with hypothermia, even if applied as a ‘bridging’ intervention, that is to say, bridging the time from onset of reperfusion until the desired level of hypothermia has been achieved. With respect to simultaneous administration, the data from the present study extend the findings from neural tissue to myocardial tissue (Fig. 2): whereas subanaesthetic xenon and mild hypothermia are not effective on their own, in combination they induce cardiac postconditioning. Between both cases of synergistic postconditioning there are a number of important differences. In the rat brain, the individual stimuli produce synergistic tissue protection if applied over a time scale of several hours after the onset of reperfusion (hypothermia after 1 h, xenon after up to 8 h).5 Even a large time gap between the component interventions (up to 5 h) does not prevent their synergistic action.5 In the case of the heart, by contrast, the window of therapeutic opportunity seems much more restricted. Although there is experimental evidence that lethal myocardial reperfusion injury progresses for many hours following the restoration of coronary blood flow,14,15 no modality capable of producing cardiac postconditioning has been shown to be efficacious after more than 30 min of reperfusion have elapsed. The restricted timescale, which cardiac postconditioning seems to demand, is reflected in our experimental protocol.
That Martin et al.5 applied mild hypothermia as the earlier intervention, followed by delayed xenon inhalation, should be noted. Their clinical vision behind this arrangement was that a human neonate who has suffered a perinatal ischaemic–hypoxic insult could receive hypothermic treatment while being transferred to a centre where xenon is available. In accordance with our clinical vision for patients with myocardial infarction or cardiopulmonary arrest, we applied both interventions in reverse chronological order. It is possible that this difference could account for the inefficiency in the asynchronous application observed in our study: asynchronous application of xenon and hypothermia does not produce a reduction in infarct size. This finding does therefore not support the hypothesis that inhalation of 20% xenon for 30 min will act synergistically with subsequent hypothermia. However, if applied simultaneously, both stimuli produce cardiac postconditioning. It remains speculative whether other combinations, for example low concentrations of xenon along with deep hypothermia or higher concentrations of xenon along with mild hypothermia, might induce infarct size reduction when applied asynchronously. In addition, the time frames in which the respective stimuli were applied might also influence myocardial protection.
A detailed investigation of the mechanisms underlying a possible protective effect was beyond the scope of this study. A heterogeneous group of cellular kinases, collectively termed ‘reperfusion injury survival kinases’ (RISKs) by Yellon and Hausenloy,14 play a pivotal role in both preconditioning and postconditioning processes. Likewise, convergence of signal transduction pathways towards inhibiting mitochondrial permeability transition is a common feature of most known cardiac preconditioning and postconditioning processes. Pagel et al.16 have demonstrated that these principles are applicable to myocardial preconditioning induced by the noble gases helium, neon and argon. It seems therefore reasonable to speculate that postconditioning induced by the synergism between xenon and hypothermia involves similar mechanisms.
From other studies we know stimuli which do not produce cardiac postconditioning by themselves, but do so in combination.17,18 In contrast, postconditioning stimuli which are efficacious on their own do not necessarily enhance their respective effects when combined.19–21 One might argue that the power of our group size was not sufficient to demonstrate significant differences between controls and low-dose xenon or mild hypothermia alone. However, in previous publications using the same experimental model significant changes were demonstrated in the same group size.19,22 It might be possible that increasing group size will reveal a significant effect of the respective interventions alone. However, the present results indicate that combination of two light stimuli can induce profound cardioprotection.
Although returning the animals onto their warming pads at the end of the hypothermic interval was the only re-warming measure, group Xe20 + Hypo34 re-warmed by 2.4°C from a starting temperature of 33.5°C. Higher rates of re-warming have the potential to accelerate functional and metabolic disturbances.23 As a result, any degree of cardioprotection might have been influenced by the re-warming period itself. We limited the period of hypothermia to 1 h, and it remains open whether longer periods of hypothermia might be even more protective against myocardial infarction.
In the present study we observed a substantial decline in HR and RPP associated with hypothermia. This implies reduced cardiac workload and suggests a more favourable cellular energy balance in the hypothermic groups. It could be argued that more plentiful cellular energy reserves might contribute to the infarct size reduction observed in group Xe20 + Hypo34. However, atrial pacing has been used in previous studies to equalize cardiac work between hypothermic animals and controls, demonstrating that the infarct sparing effect of hypothermia is still evident when the decline in HR was compensated for.24 Other investigators support the notion that infarct size does not correlate with RPP during early reperfusion.25 We therefore conclude that the cardioprotection observed in group Xe20 + Hypo34 reflects a pharmacodynamic effect of the combination of xenon and hypothermia, rather than being a consequence of reduced cardiac workload during reperfusion.
In summary, in the present study we demonstrated that the combination of xenon in low concentration (20%) and mild hypothermia (34°C) produces cardiac postconditioning in a rat model, whereas asynchronous administration of first xenon then hypothermia was not effective to significantly reduce infarct size in our model. However, a prerequisite for this approach to be clinically effective would appear to be the timely institution of hypothermia. In humans, the latter requires technical proficiency and careful procedural planning, which is why reperfusion after percutaneous coronary interventions following acute myocardial infarction is likely to provide a more suitable setting for clinical studies than the largely more unpredictable postcardiac arrest scenario.
The study was funded in part by an MD-medical research trainee (AGIKO) grant (92003450) to A.H. from the Netherlands Organization for Health Research and Development (ZonMw).
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