Effects of halothane, enflurane, isoflurane, sevoflurane and desflurane on myocardial reperfusion injury in the isolated rat heart

Transcription

1 British Journal of Anaesthesia 1998; 81: Effects of halothane, enflurane, isoflurane, sevoflurane and desflurane on myocardial reperfusion injury in the isolated rat heart W. SCHLACK, B. PRECKEL, D. STUNNECK AND V. THÄMER Summary A specific action against myocardial reperfusion injury of the oxygen paradox type was recently characterized for halothane after anoxic perfusion in isolated rat hearts and isolated cardiomyocytes. In this study, we have characterized the protective effects of the clinically available inhalation anaesthetics during reperfusion after ischaemia. In isolated, isovolumically beating rat hearts perfused at a constant flow (10 ml min 1, PO2 80 kpa) and paced at 350 beat min 1, we determined left ventricular developed pressure (LVDP) and release of creatine kinase (CKR) as indices of myocardial performance and cellular injury, respectively. Seven control hearts underwent 30 min of no-flow ischaemia and 1 h of reperfusion. In the treatment groups, halothane, enflurane, isoflurane, sevoflurane or desflurane (each group n 6) was added to the perfusion medium for the first 30 min of reperfusion at a concentration corresponding to 1.5 MAC in the rat. In the control group, cellular injury occurred at early reperfusion (peak CKR 283 (SEM 57) iu litre 1 at 10 min of reperfusion). Peak CKR to the coronary venous effluent was attenuated by all anaesthetics (halothane group 156 (45), enflurane group 134 (20), sevoflurane group 132 (20), desflurane group 159 (25) iu litre 1 ; each P 0.05). Isoflurane did not differ from controls (303 (53) iu litre 1 ; P 0.5). In the sevoflurane group, there was a delayed peak CKR after discontinuation of the anaesthetic at 30 min of reperfusion (260 (34) iu litre 1 ). Functional recovery was improved by all anaesthetics, but was seen much earlier with desflurane (LVDP 28 (3)% of baseline at 5 min reperfusion compared with halothane (6 (1)%), enflurane (11 (3)%), isoflurane (9 (6)%), sevoflurane (10(2)%) and controls (3 (1)% of baseline)). At 30 min of reperfusion, recovery of LVDP was improved to a similar extent by all anaesthetics (halothane 30 (9)%, enflurane 36 (9)%, isoflurane 33 (5)%, sevoflurane 30 (5)%, desflurane 36 (4)% of baseline values) compared with controls (13 (5)%; each P 0.05). All inhalation anaesthetics protected against myocardial reperfusion injury, but showed differences in attenuation of cellular injury and functional recovery. These differences may suggest different protective mechanisms. (Br. J. Anaesth. 1998; 81: ). Keywords: anaesthetics volatile, halothane; anaesthetics volatile, enflurane; anaesthetics volatile, isoflurane; anaesthetics volatile, sevoflurane; anaesthetics volatile, desflurane; heart, myocardial function; model, heart; rat It was reported recently that cardioprotection by halothane in isolated anoxic reoxygenated rat hearts was much more pronounced if the substance was administered after the period of anoxic perfusion (during reoxygenation) than if given during the anoxic period itself. 1 From these findings it was concluded that, apart from displaying anti-ischaemic effects, halothane may have a specific effect on reperfusion injury. This protective effect was also confirmed in vivo in a rabbit model of coronary artery occlusion and subsequent reperfusion. 2 One MAC of halothane given during the initial 15 min of reperfusion caused a 38% reduction in infarct size, even if the haemodynamic effects were counteracted by i.v. norepinephrine (noradrenaline). Using the model of anoxic reoxygenated isolated cardiomyocytes, it was possible to confirm a protective effect against reperfusion injury of the oxygen paradox type and to study the underlying protective mechanism. 3 5 Halothane 1.5 MAC given during reoxygenation prevented reoxygenation-induced intracellular Ca 2 oscillations which are responsible for cellular hypercontracture and cell death at early reperfusion. 6 Therefore, the above cited studies strongly suggest a specific protective mechanism of halothane against reperfusion injury in the heart. We hypothesized that other inhalation anaesthetics may have similar effects on the ischaemic reperfused myocardium. Therefore, the aim of this study was to describe the effects of the currently available inhalation anaesthetics, halothane, enflurane, isoflurane, sevoflurane and desflurane, on myocardial reperfusion injury after ischaemia. We used an isolated rat heart model with 30 min of no-flow ischaemia and 1 h of reperfusion and supplied the inhalation anaesthetics for the first 30 min of reperfusion. In this model, extrinsic humoral and autonomic nervous system influences were excluded and ventricular volume, coronary flow and heart rate were kept constant. Materials and methods The study was performed in accordance with the regulations of the German Law for the Protection of Animals and local institutional regulations. W. SCHLACK, MD, DEAA B. PRECKEL, MD (Institut für Klinische Anaesthesiologie); D. STUNNECK, CAND. MD, V. THÄMER, MD (Physiologisches Institut I); Heinrich-Heine-Universität Düsseldorf, Postfach , D Düsseldorf, Germany. Accepted for publication: May 8, Correspondence to W. S.

2 914 British Journal of Anaesthesia EXPERIMENTAL PREPARATION Hearts from male Wistar rats (weighing g), anaesthetized with halothane, were excised rapidly and mounted on a Langendorff perfusion system. Retrograde perfusion was initiated with an oxygenated modified Krebs Henseleit buffer containing (mmol litre 1 ): NaCl 116, KCl 4.7, MgSO 4 1.1, KH 2 PO , NaHCO , CaCl , glucose 8.3 and pyruvate 2.0; gassed with 95% oxygen 5% carbon dioxide. Flow in the system was controlled by two calibrated roller pumps (Model 7518, Cole- Parmer Instruments, IL, USA). After ligating both venae cavae, the right ventricle was vented via the pulmonary artery with a Teflon catheter (1.2 mm od). Heart rate was maintained at 350 beat min 1 by atrial pacing. The stimulation voltage was maintained at 20% above threshold (control 2 4 V) and adjusted continuously throughout the experiment (up to 12 V in reperfused hearts, if necessary). After completion of the experimental preparation, the heart was placed in a water-jacketed chamber at 37 C, filled with humidified, warmed air. The hearts were perfused at a flow rate of 10 ml min 1 which was kept constant throughout the perfusion period. During ischaemia, the heart chamber was filled with normal saline to maintain heart temperature at 37 C and gassed with nitrogen to prevent oxygen supply to the ischaemic myocardium by diffusion. In order to ensure the presence of the inhalation anaesthetic with the onset of reperfusion, the hearts were perfused for the last 2 min of the ischaemic period with anoxic buffer equilibrated with 95% nitrogen 5% carbon dioxide containing the different inhalation anaesthetics in the concentrations specified below or buffer alone (controls) at a flow rate of 2 ml min 1. Reperfusion was then initiated by switching back to the initial normoxic perfusion conditions. For administration of 1.5 MAC of halothane, enflurane and isoflurane, a saturated solution was prepared in the perfusion buffer at 22 C and infused into the perfusion system near the aortic cannula at appropriate fractions of total coronary flow in order to achieve final concentrations of 0.45, 0.96 and 0.47 mmol litre 1, respectively, using a calibrated syringe pump (Model 5003, Precidior Infors, Basel, Switzerland). For the 2-min anoxic perfusion, the saturated solution was prepared in the anoxic buffer. For sevoflurane administration, the saturated solution was prepared using distilled water. During administration of sevoflurane, a more concentrated buffer was used so that the final concentration of solutes was not altered and the final sevoflurane concentration was 0.71 mmol litre 1. Only glass or Teflon tubing was in contact with the perfusion medium containing inhalation anaesthetics. Desflurane was supplied to the perfusion medium using a calibrated vaporizer (Devapor, Dräger Werke, Lübeck, Germany) using a hollow fibre oxygenator (D705 Midflow, DIDECO, Mirandola, Italy) to achieve a perfusate concentration of 1.0 mmol litre 1. Concentrations of the inhalation anaesthetics were determined in the saturated solutions by gas chromatography. Haemodynamic measurements For measurements of left ventricular pressure, a latex balloon (size No. 4, Hugo Sachs Elektronik, March, Germany) was introduced into the left ventricle via the cut mitral valve. The balloon was fixed at the tip of a stainless steel cannula (length 5.9 cm) which was connected directly to a pressure transducer (Gould P23, Cleveland, OH, USA). Left ventricular pressure, and (by electronic differentiation) the velocity of the change in left ventricular pressure (dp/dt) were monitored continuously on an ink recorder (Gould, Mark 260, Cleveland, OH, USA). At the beginning of each experiment, the latex balloon was filled with normal saline (air-bubble free) to achieve an enddiastolic left ventricular pressure of 5 mm Hg. This volume was then held constant for the rest of the experiment. Left ventricular pressure and coronary perfusion pressure signals were digitized at a sampling rate of 2000 Hz using an analogue to digital converter (Data Translation 2801, Marlboro, MA, USA) and then processed on a personal computer system. Left ventricular end-diastole was determined as the point when left ventricular dp/dt started its rapid upstroke after crossing the zero line. Left ventricular end-systole was defined as the point of minimum dp/dt. 7 Left ventricular end-diastolic pressure (LVEDP), developed pressure (LVDP), maximum and minimum dp/dt (dp/dtmax and dp/dtmin) were obtained from the digitized signals. Only hearts with an LVDP greater than 70 mm Hg during the initial control period were used for the study. Metabolic measurements Aliquots from the perfusion medium and the coronary venous effluent perfusate were sampled anaerobically at the times indicated. Samples were processed immediately for PO 2 measurements (ABL 30, Radiometer, Copenhagen, Denmark). Oxygen consumption (VO 2 ) was calculated according to Fick s principle with the use of Bunsen s solubility coefficient ( l mm Hg 1 ml 1 ) at 37 C as follows: 1 V! O 2( l min ) = ( PaO Pv 2 O ) CF 2 where PaO arterial PO 2 2, P vo venous PO 2 2 (kpa) and CF coronary flow (ml min 1 ). For determination of creatine kinase (CK) release, 1-min samples of the effluent were collected at the times indicated (fig. 1). CK activity was measured with an optimized standard method according to the recommendations of the Deutsche Gesellschaft für Klinische Chemie (CK test, Boehringer, Mannheim, Germany). EXPERIMENTAL PROGRAMME After mounting the heart on the Langendorff apparatus, a preparation and stabilization period of 30 min was allowed. The experimental procedure consisted of three phases: baseline, ischaemia and reperfusion (fig. 1). After a 15-min baseline period with steady state conditions and control measurements of all experimental variables, the hearts underwent 30 min of no-flow ischaemia. After the ischaemic period, the hearts were reperfused with the initial oxygenated medium. The inhalation anaesthetics were added to the perfusion medium to achieve concentrations corresponding to 1.5 rat MAC during the first 30 min of reperfusion starting with a 2-min anoxic wash-in of

3 Inhalation anaesthetics and reperfusion injury in vitro 915 the agents (each n 6). Seven hearts served as controls and underwent the ischaemia reperfusion programme without intervention. Five hearts received the different inhalation anaesthetics (except halothane) in a randomized order for 5 min during normoxic perfusion conditions for assessment of the haemodynamic effects on normal myocardium in this experimental model. After each agent, 15 min were allowed for full recovery of all haemodynamic variables. The data for halothane on normal myocardium of the rat were taken from a previous study. 1 DATA ANALYSIS Data are presented as mean (SEM). The effects of inhalation anaesthetics on normal myocardium were assessed by Student s t test for paired observations. In the ischaemia reperfusion experiments, statistical analysis was performed using two-way analysis of variance (ANOVA) for time and treatment (experimental group) effects. If an overall significant difference between groups was found, a comparison was performed for each time using one-way ANOVA followed by Dunnetts s post-test when appropriate. Table 1 Haemodynamic variables. Effect of the inhalation anaesthetics on normal myocardium. Data are mean (SEM), n 5. LVDP Left ventricular developed pressure; dp/dtmax maximum velocity of the change in left ventricular pressure; VO 2 myocardial oxygen consumption. *P 0.05, **P 0.01, ***P compared with baselines Baseline 1.5 MAC LVDP (mm Hg) Halothane 99.3 (1.8) 52.0 (2.7)*** Isoflurane 79.8 (13.5) 51.7 (10.2)** Enflurane 91.9 (6.9) 64.1 (4.4)*** Sevoflurane 92.6 (5.5) 59.8 (7.0)** Desflurane 93.5 (8.0) 70.7 (6.6)* dp/dtmax (mm Hg s 1 ) Halothane 3465 (78) 2344 (78) Isoflurane 2689 (390) 1996 (373)** Enflurane 3046 (167) 2300 (169)*** Sevoflurane 3014 (124) 2053 (235)** Desflurane 3172 (153) 2519 (145)** VO 2 ( l min 1 ) Halothane (29.5) (21.0) Isoflurane (15.1) (12.4)*** Enflurane (16.5) (18.1)*** Sevoflurane (5.7) (7.2)*** Desflurane (4.4) (4.5)** Results A total of 54 hearts were used. Fourteen hearts did not fulfill the predefined quality criteria (LVDP 70 mm Hg) and were excluded. HAEMODYNAMIC FUNCTION Basal values and effects of the inhalation anaesthetics on normal myocardium In five hearts, enflurane, isoflurane, sevoflurane and desflurane were given in a randomized order at a concentration corresponding to 1.5 MAC (measurements were made after 5 min under steady state conditions and 15 min were allowed for recovery between interventions). Data for halothane (1.5 MAC) were obtained from a previous study with measurements made after 15 min of steady state conditions. 1 During desflurane administration, there was a small initial increase in LVDP and dp/dt of 10 20% before these variables started to decline. This initial increase in contractile force was observed only with desflurane. All inhalation anaesthetics reduced LVDP (halothane to 52%, enflurane to 70%, isoflurane to 65%, sevoflurane to 65% and desflurane to 76% of baseline values, respectively) and dp/dtmax (halothane to 68%, enflurane to 76%, isoflurane to 74%, sevoflurane to 68% and desflurane to 79% of baseline values, respectively). Simultaneously, VO 2 was reduced (to a similar extent with halothane (to 86%), enflurane (to 75%), isoflurane (to 69%) and sevoflurane (to 76%) but was less pronounced with desflurane (to 90% of baseline)) (table 1). End-diastolic pressure during ischaemia reperfusion During ischaemia, LVEDP increased progressively in all groups from 5.1 (0.3) mm Hg during normoxic baseline conditions to 54.9 (3.0) mm Hg at the end of the ischaemic period, indicating the development of ischaemic contracture. Initiation of anoxic perfusion for wash-in of the inhalation anaesthetics before reperfusion had no effect on LVEDP. With the onset of reperfusion, all groups showed a further increase in LVEDP (reperfusion contracture, table 2). All inhalation anaesthetics moderately improved overall LVEDP recovery during reperfusion compared with controls (P 0.05 each; ANOVA), indicating a reduction in reperfusion contracture. After 60 min of reperfusion, LVEDP had recovered in all groups to similar values. Recovery of contractile function during reperfusion Figure 1 (top) shows recovery of LVDP as an index of contractile function. During baseline conditions, LVDP was similar in all groups. During reperfusion after ischaemia, recovery of LVDP was very poor in the control group, reaching only 13 (2)% of baseline values at 60 min of reperfusion. Administration of all inhalation anaesthetics resulted in improved functional recovery that was similar for all agents at 60 min of reperfusion (33 (3)% of baseline; P 0.05). However, the time course of recovery was different for the agents: halothane, enflurane and isoflurane showed a similar pattern of recovery (fig. 1, top left), starting with a gradual recovery of LVDP during the initial 30 min of reperfusion when the inhalation anaesthetics were present, followed by a further increase in LVDP by 18% after anaesthetic withdrawal. Sevoflurane showed a similar pattern of recovery during the first 30 min of reperfusion, but there was no further functional improvement after its withdrawal (fig. 1, top right). Desflurane showed a different pattern, reaching the final state of functional recovery within the first 2 min of reperfusion. Recovery of dp/dtmax as an index of myocardial contractility was parallelled by recovery of LVDP (table 2).

4 916 British Journal of Anaesthesia Table 2 Haemodynamic variables during the ischaemia reperfusion experiments. Data are mean (SEM), n 6. CPP Coronary perfusion pressure; LVEDP left ventricular end-diastolic developed pressure; dp/dtmax maximum velocity of the change in left ventricular pressure; VO 2 myocardial oxygen consumption. *P 0.05 compared with controls; P 0.05 compared with baseline Inhalation anaesthetic Reperfusion Baseline 5 min 25 min 30 min 60 min CPP (mm Hg) Control 87.1 (10.5) (4.6) (7.6) (7.8) (9.4) Halothane 75.2 (8.3) 93.9 (7.0) 97.2 (7.4) (8.0) (7.5) Isoflurane 72.4 (10.3) (5.1) (5.7) (5.3) (6.7) Enflurane 75.7 (9.7) 97.0 (6.0) 99.1 (7.9) 97.3 (8.0) (10.3) Sevoflurane 74.4 (9.2) (4.0) (15.6) (14.7) (16.4) Desflurane 81.0 (17.3) 83.0 (3.5)* 98.7 (8.6) (10.0) (13.7) LVEDP (mm Hg) Control 4.9 (0.5) (5.7) 89.4 (4.1) 85.6 (4.1) 78.8 (4.5) Halothane 4.9 (0.5) (7.7) 81.4 (7.1) 76.6 (8.9) 83.9 (7.6) Isoflurane 5.5 (0.7) (10.1) 72.0 (6.4) 67.2 (6.2) 72.3 (3.3) Enflurane 4.5 (0.4) (7.8) 67.0 (5.6)* 59.1 (6.4)* 68.8 (8.0) Sevoflurane 4.0 (0.7) (8.5) 68.4 (4.9) 70.0 (2.6) 60.7 (4.1) Desflurane 5.5 (0.5) (4.4)* 71.4 (3.1) 74.3 (4.1) 77.7 (8.2) dp/dtmax (mm Hg s 1 ) Control 3617 (296) 680 (90) 839 (68) 956 (96) 1012 (101) Halothane 3237 (356) 804 (147) 1300 (172) 1717 (381) 1339 (232) Isoflurane 3115 (183) 667 (111) 1218 (243) 1474 (244) 1210 (134) Enflurane 3054 (174) 670 (60) 1265 (157) 1730 (300) 1378 (248) Sevoflurane 3181 (250) 661 (62) 1284 (147) 1386 (184) 1340 (135) Desflurane 3092 (188) 1288 (75)* 1754 (127)* 1733 (64) 1457 (110) VO 2 ( g min 1 ) Control (9.5) (8.6) (14.1) (14.6) (17.3) Halothane (13.1) (15.5) (6.5) (5.9) (9.1) Isoflurane (6.8) (11.4) (8.7) (6.8) (6.4) Enflurane (11.9) (5.6) (16.8) (9.2) (7.7) Sevoflurane (7.3) (13.2) (17.5) (14.9)* (14.6) Desflurane (9.3) (6.9) (11.7) (13.7) (18.7) Figure 1 Time course of left ventricular developed pressure (LVDP) as an index of myocardial function and creatine kinase release (CKR) as an index of cellular injury. Controls are the same on the left and right side of the figure. Drug time when 1.5 MAC of the respective inhalation anaesthetic was added to the perfusion medium. Coronary vascular resistance during reperfusion CPP is a direct measure of coronary vascular resistance in this experimental model because coronary flow was kept constant (table 2). With the onset of reperfusion, CPP increased to 130% of baseline in the control group. This initial increase in CPP after ischaemia was similar in the presence of halothane (to 125%), enflurane (to 128%), isoflurane (to 140%) and sevoflurane (to 143%) and was absent in the presence of desflurane (to 102%, P 0.05 vs control). In all groups, there was a further progressive increase in CPP during the reperfusion period which was most pronounced in the sevoflurane group.

5 Inhalation anaesthetics and reperfusion injury in vitro 917 METABOLIC MEASUREMENTS Creatine kinase CK activity in the coronary venous effluent was determined as an index of cellular injury (fig. 1, bottom). In the control group, CKR increased rapidly during the early reperfusion period with a peak value of 283 iu litre 1 at 10 min of reperfusion. This initial peak CKR was markedly attenuated by halothane, enflurane, sevoflurane and desflurane (to 42%, 45%, 47% and 39% of the peak CKR in the untreated controls). Isoflurane had no effect on initial cellular injury and peak CKR was similar to that of controls (107% of controls). Although sevoflurane markedly attenuated the early peak CKR, a delayed peak CKR occurred immediately after removal. Oxygen consumption During baseline conditions, VO 2 was not different between groups. With the onset of reperfusion, VO 2 of the reperfused myocardium was as high as during baseline conditions in all groups, despite the markedly reduced contractile state. In the control group, VO 2 then rapidly declined during reperfusion to 56% of baseline at the end of the reperfusion period (P 0.05). In the groups treated with inhalation anaesthetics, VO 2 during reperfusion tended to be higher than in untreated controls, but also declined during reperfusion in the halothane (to 78%), isoflurane (to 72%) and enflurane groups (to 86% of baseline at 60 min of reperfusion). VO 2 remained at the pre-ischaemic baseline level in the sevoflurane group (97% of baseline at 60 min of reperfusion) (table 2). Discussion The main finding of our study was that all inhalation anaesthetics offered some protective effect against reperfusion injury in the heart. However, there were differences in the effects on cellular injury (which was attenuated by all anaesthetics except isoflurane) and functional recovery (which was improved by all agents to a similar degree, but with a different time course). These differences suggest different protective mechanisms, which have to date been characterized at a cellular level for only halothane. critique of methods Five hearts received four different inhalation anaesthetics each for assessment of the haemodynamic effects on normal myocardium in this experimental preparation. Although these interventions were made in a randomized order, effects of previous exposure to a different anaesthetic cannot be excluded. However, the data confirm a myocardial depressant effect of all anaesthetics in this preparation. Our study used an isolated buffer perfused heart model, which has the advantage of excluding most haemodynamic and humoral side effects of inhalation anaesthetics. But a myocardial depressant effect of the anaesthetics was observed in the present model and was shown by a reduction in LVDP and dp/dtmax in non-ischaemic hearts. However, during reperfusion, LVDP was higher in most of the treat- ment groups compared with controls, which makes it unlikely that changes in ventricular pressure were responsible for the protective effect. While the experimental model of our study was designed to assess direct myocardial effects of inhalation anaesthetics on reperfusion injury, at the same time it excluded several mechanisms that may also be important in the in vivo situation of ischaemia and reperfusion, such as changes in global haemodynamics and sympathetic nervous system activity, and activation of neutrophils in reperfused myocardium (for review see Mullane and Young 8 ). Consequently, the effect of the inhalation anaesthetics in vivo may be different from what was observed in our study. For halothane, isoflurane and sevoflurane it has been shown that they can reduce post-ischaemic accumulation of neutrophils in reperfused myocardium, 9 which may be an important protective mechanism in vivo. As the danger of impeding hypercontracture in the reoxygenated myocardium is evoked by reactivation of mitochondrial energy production, 10 a protective agent should be present in the myocardium at the onset of reoxygenation. Therefore, we started the supply of the inhalation anaesthetics with a 2-min anoxic wash-in before reperfusion with the oxygenated buffer. During anoxic perfusion there was no change in LVEDP as an index of myocardial contracture, while LVEDP increased immediately with the onset of reperfusion with oxygenated buffer. In a previous study we have shown that during anoxic perfusion, there is no significant CK release, indicating the absence of lethal cellular injury at this time. 1 Thus it is unlikely that this short period of anoxic perfusion itself had an effect on ischaemia reperfusion injury in this study. In addition, the control hearts also underwent a similar anoxic perfusion programme as the treatment hearts. INTERPRETATION OF RESULTS Reperfusion of myocardium after a prolonged period of ischaemia initiates cellular and biochemical changes which reduce the amount of potentially salvageable myocardium and impair the contractile function of the surviving myocardium ( reperfusion injury ). The term lethal reperfusion injury was introduced for the irreversible deterioration of myocardium that can be avoided by modifications of the conditions of reperfusion. 11 If the preceding ischaemia is less severe, reperfusion results in a reversible state of non-lethally injured myocardium showing contractile dysfunction: myocardial stunning. 12 For halothane, a specific protective effect against reperfusion injury had been demonstrated that can be separated from its anti-ischaemic effects. 1 Several studies have described protective effects of enflurane and isoflurane in ischaemia reperfusion situations: enflurane and isoflurane improved post-ischaemic functional recovery of isolated hearts after cardioplegic arrest, 13 after hypoxic perfusion (isoflurane) 14, after global ischaemia (enflurane, 15 isoflurane) 16 and after 1-day hypothermic preservation (isoflurane). 17 In vivo, attenuation of myocardial stunning was found for isoflurane in dogs However, from these studies, it was not possible to distinguish antiischaemic effects from effects on myocardial reperfusion injury because in these studies the inhalation

6 918 British Journal of Anaesthesia anaesthetics were given before the onset of ischaemia or during the ischaemic period. In the in vivo studies, the haemodynamic effects of the inhalation anaesthetics (i.e. a reduction in sympathetic tone, myocardial inotropy and myocardial work) may also have contributed to the observed protective effects. For sevoflurane and desflurane, only anti-ischaemic effects have been studied to date, but there are no studies investigating the effects of these agents during reperfusion. Therefore, in this study we used isolated hearts where systemic haemodynamic effects are absent and where the substances were given selectively during the initial reperfusion period only. In a previous study we used a similar isolated heart model for demonstration of a pronounced protective effect against reperfusion injury by halothane. 1 In that study, ischaemia was simulated by anoxic perfusion in order to administer halothane during ischaemia also. However, anoxic perfusion partially eliminates the metabolic waste from the ischaemic myocardium and may thereby modify ischaemia reperfusion injury. In this study, we used no-flow ischaemia followed by reperfusion, which comes closer to the in vivo situation of ischaemia reperfusion. Our previous study showed that halothane reduced lethal cell damage during reperfusion and led to improved functional recovery. These findings were confirmed in our study. We expected that the other volatile anaesthetics would protect against reperfusion injury in a similar manner. Surprisingly, they had different effects when given during reperfusion: only enflurane was comparable with halothane; during desflurane administration, functional recovery occurred more rapidly; isoflurane had no effect on lethal cell damage but improved functional recovery; and with sevoflurane, lethal cell damage was delayed and occurred after discontinuation of the anaesthetic. However, functional recovery with sevoflurane was similar to that seen in the presence of halothane, enflurane or isoflurane. Recovery of contractile function In experimental models such as ours, functional recovery depends mainly on two factors. 23 First, the amount of lethally injured myocardium can limit the extent of functional recovery by a reduction in contractile mass. Second, even after short periods of ischaemia, contractile function of non-lethally injured myocardium is depressed and may need several hours or days for full recovery. This phenomenon is termed myocardial stunning. Consequently, the effect of inhalation anaesthetics on recovery of myocardial function may be explained by one or both of these mechanisms. In the desflurane group, a transient positive inotropic effect may have contributed to the time course of functional recovery: only during desflurane administration was there rapid improvement in contractility within the first minutes of reperfusion to close to the final state of functional recovery. Desflurane also showed a small initial positive inotropic effect in the normoxic control hearts, which is most likely caused by direct catecholamine release from autonomic nerve endings. 24 It is well known that stunned myocardium responds well to inotropic stimulation, 25 and this initial positive inotropic effect of desflurane may be responsible for the initial rapid improvement in contractile function in this group. Lethal cellular injury With the onset of reperfusion, VO 2 was high in the control hearts and then declined rapidly during the initial reperfusion period, while simultaneously, CK was released from the myocardium indicating the occurence of lethal reperfusion injury during this time. Isoflurane was the only inhalation anaesthetic that had no effect on CKR during reperfusion or on VO 2 compared with controls. Sevoflurane delayed CKR until its discontinuation. The lack of a protective effect of isoflurane against lethal cell damage was also observed in isolated working rat hearts (where halothane or isoflurane were given also before ischaemia and during reperfusion) and in vivo in rabbits, where isoflurane failed to reduce myocardial infarct size after coronary occlusion and reperfusion The finding of improved functional recovery of post-ischaemic myocardium in the presence of isoflurane is in accordance with two previous in vivo studies that also found attenuation of myocardial stunning in non-lethally injured myocardium The mechanisms leading to cell death and myocardial stunning may differ slightly (for review see Hearse and Bolli 23 ). Therefore, the difference in protective effects against lethal cell damage and myocardial stunning also suggests different protective effects of inhalation anaesthetics given during reperfusion against these two forms of myocardial reperfusion injury. The concept of early lethal reperfusion injury was developed from investigation of isolated anoxic reoxygenated heart cells. 28 Here it was shown that the critical phase for development of early lethal reperfusion injury is the first minutes after resupply of oxygen, when intracellular calcium overload 29 and SR dysfunction 6 may trigger cellular hypercontracture and cell death. From these findings, it was concluded that a protective agent could be discontinued after this critical time. 28 This concept of early discontinuation of the protective substances worked in our experimental model for halothane, enflurane and desflurane, but not for sevoflurane, which showed a delayed peak CKR after discontinuation, indicating the occurrence of lethal cellular injury at this time. However, the total amount of CK released from the reperfused myocardium was also reduced by sevoflurane. The reason for these differences is unknown but suggests a different action of sevoflurane on postischaemic myocardium. For halothane, the protective mechanism against lethal reperfusion damage of the oxygen paradox type was recently identified 3 4 : halothane prevented the rapid Ca 2 cycling of the sarcoplasmic reticulum (SR) that is found during reoxygenation after a severe ischaemic insult. These Ca 2 oscillations occur at the calcium release channel (ryanodine receptor) of the SR and are responsible for cellular hypercontracture and cell death during early reperfusion. 6 Da ta from non-ischa emic reperfused isolated heart cells showed that halothane and enflurane but not isoflurane act on this calcium release channel of the SR. 30 The finding that isoflurane does not influence SR calcium release channel may explain the failure of

7 Inhalation anaesthetics and reperfusion injury in vitro 919 isoflurane to protect against early lethal reperfusion injury. The action of sevoflurane on this channel is still unknown. However, except for halothane, the mechanisms of myocardial protection against lethal reperfusion injury by inhalation anaesthetics have not been investigated. They do not seem to be related to the negative inotropic effects of the agents; both halothane, which has the most pronounced negative inotropic effects in isolated hearts during normoxia (this study) or during post-ischaemic periods, 21 and desflurane, which has the lowest negative inotropic effects, were comparable with regard to myocardial protection. This example also argues against a simple relationship between the protective effect and physicochemical properties (such as lipid solubility, which is lowest for desflurane and highest for halothane but both offered similar protection, and intermediate for isoflurane which had no protective effect against lethal cell injury). In summary, the results of our study provide some evidence that inhalation anaesthetics have different mechanisms of protection against reperfusion injury, but further experimental work is needed to clarify these mechanisms at the cellular level. Acknowledgement We thank Ms Elke Hauschildt, BTA, and Mr. Jörg Stunneck, CAND MD, for technical assistance, and Mr Husmann, Max-Planck Institute for Carbon Research, Mühlheim, Germany, for measurements of the inhalation anaesthetics. This work is part of the MD Thesis of D.S. References 1. Schlack W, Hollmann M, Stunneck J, Thamer V. Effect of halothane on myocardial reoxygenation injury in the isolated rat heart. 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