Halothane reduces reperfusion injury after regional ischaemia in the rabbit heart in vivo

Transcription

1 British Journal of Anaesthesia 1997; 79: Halothane reduces reperfusion injury after regional ischaemia in the rabbit heart in vivo W. SCHLACK, B. PRECKEL, H. BARTHEL, D. OBAL AND V. THÄMER Summary In addition to having anti-ischaemic effects, halothane can protect isolated rat hearts and isolated cardiomyocytes against reperfusion injury of the oxygen paradox type. The aim of this study was to investigate if halothane can also protect against myocardial reperfusion injury in vivo. Twenty-two rabbits anaesthetized with -chloralose underwent 30 min of occlusion of a major coronary artery and 2 h of subsequent reperfusion. Seven animals received 1 MAC of halothane for the first 15 min of reperfusion (halothane group), and eight animals served as untreated controls (control group). In seven additional animals, the haemodynamic effects of halothane were antagonized by an i.v. infusion of noradrenaline (halothane noradrenaline group). We measured cardiac output (CO) by an ultrasonic flow probe around the ascending aorta, left ventricular pressure (LVP) by a tip manometer and infarct size by triphenyltetrazolium staining. Baseline LVP was mean 92 (SEM 4) mm Hg and CO was 289 (16) ml min 1. During coronary occlusion, LVP was reduced to 86 (4) % of baseline and CO to 84 (4)% (similar in all groups). During halothane administration at reperfusion, LVP declined further to 55 (6) % of baseline and CO to 66 (9) % (P 0.05 halothane group vs control group). Noradrenaline prevented the reduction in LVP (halothane noradrenaline group 87 (5) % of baseline, control group 84 (6) %) and reduction in CO (halothane noradrenaline group 89 (5) %, control group 83 (6) %). Infarct size was 49 (6) % of the area at risk in controls and was reduced markedly by administration of halothane to 32 (3) % in the halothane group (P 0.05) and to 30 (3) % in the halothane noradrenaline group (P 0.05). Treatment with halothane during the early reperfusion period after myocardial ischaemia protected the myocardium against infarction in vivo, independent of the haemodynamic effects of halothane. (Br. J. Anaesth. 1997; 79: 88 96). Key words Anaesthetics volatile, halothane. Heart, ischaemia. Heart, reperfusion injury. Heart, myocardial function. Model, rabbit. Rabbit. The anti-ischaemic effects of halothane were reported as early as 1969 by Spieckermann and colleagues 1 for dog hearts during normothermic ischaemia, and a protective effect of halothane against ischaemic injury was confirmed in several experimental studies. 2 7 Recently, we have shown in isolated rat hearts that cardiac protection by halothane was more pronounced if it was administered after ischaemia during reperfusion than if it was given during the ischaemic period itself, 8 providing some evidence for a protective effect of halothane against the mechanisms of reperfusion injury. Using isolated anoxic reoxygenated cardiomyocytes, it was possible to confirm a protective effect against lethal reperfusion injury of the oxygen paradox type and to identify a protective mechanism of halothane against this type of reperfusion injury Halothane 1.5 MAC, given during reoxygenation, prevented reoxygenation-induced Ca 2 oscillations at the Ca 2 - dependent Ca 2 -release channel of the sarcoplasmic reticulum. These Ca 2 oscillations are responsible for cellular hypercontracture and cell death at early reperfusion. 11 However, a specific protective action of halothane against reperfusion injury is evident only from in vitro studies that investigated reperfusion injury of the oxygen paradox type. In vivo, the pathophysiology of reperfusion injury is more complex. It is possible that several mechanisms of lethal reperfusion injury may exist, the oxygen paradox being but one; other mechanisms may involve free radicals, activated leucocytes and recurrent ischaemia by vascular obstruction. 12 In this study, the protective effect of halothane against lethal reperfusion injury was investigated in an in vivo animal model with coronary artery occlusion and subsequent reperfusion. Previous in vivo studies have found a reduction in myocardial injury under ischaemia reperfusion conditions in the presence of halothane However, from these studies, it was not possible to distinguish between anti-ischaemic effects and effects against reperfusion injury, because halothane was given before the onset WOLFGANG SCHLACK*, MD, DEAA, BENEDIKT PRECKEL, MD, HOLGER BARTHEL, CAND MD, DETLEF OBAL, CAND MD, VOLKER THÄMER, MD, Institut für Klinische Anaesthesiologie and Physiologisches Institut I, Heinrich-Heine-Universität Düsseldorf, Düsseldorf, Germany. Accepted for publication: March 10, *Address for correspondence: Institut für Klinische Anaesthesiologie, Heinrich-Heine-Universität Düsseldorf, Postfach , D Düsseldorf, Germany.

2 Halothane and reperfusion injury in vivo 89 of ischaemia and during ischaemia. It is known that other negative inotropic drugs (e.g. calcium antagonists or beta blockers ) have potent antiischaemic effects when given before or during ischaemia. In this study, we administered halothane only during the initial reperfusion period (15 min). Halothane is known to reduce sympathoadrenergic activity, myocardial inotropy and ventricular loading. To investigate if these haemodynamic effects may be responsible for a myocardial protective effect of halothane in vivo, arterial pressure was kept stable in one experimental group by continuous infusion of noradrenaline during administration of halothane. Materials and methods The study was performed in accordance with the regulations of the German Animal Protection Law and after obtaining permission from the Animal Care Committee of the District of Düsseldorf. ANIMAL PREPARATION After local anaesthesia with EMLA cream, a marginal ear vein was cannulated in 29 New Zealand White rabbits of both sexes, weighing (mean 3.8) kg. The a nima ls were a na esthetized with sodium thiopentone mg kg 1 i.v. followed by -chloralose 80 mg kg 1 i.v. The trachea was intubated with a Woodbridge tube (3.5 mm id) and ventilation was controlled using a Starling pump (Type 874/052, Braun Melsungen, Germany). Ventilatory frequency was set at bpm and tidal volume at ml in order to maintain endexpiratory PCO 2 at approximately 4.4 kpa (Datex Capnomac Ultima, Division of Instrumentarium Corp., Helsinki, Finland). Arterial blood-gas tensions, haemoglobin concentration and packed cell volume were assessed at regular intervals (before surgery, before the study and at the end of the study). Surface electrocardiogram (ECG, Siemens- Elema AB, Medicins and Technik, Selna, Sweden) was recorded continuously. Anaesthesia was maintained by continuous infusion of -chloralose 40 mg kg 1 h 1. Additional bolus doses were given as needed during surgery. Adequacy of this anaesthesia regimen was demonstrated by lack of muscle movement and haemodynamic responses during surgery. For measurement of aortic pressure (AOP), a 20- gauge Teflon catheter was advanced from the left carotid artery into the aortic arch and connected to a Statham transducer (PD23, Gould, Cleveland, OH, USA). After cannulation of the external jugular vein, animals received a continuous infusion of normal saline 15 ml kg 1 h 1 to compensate for fluid losses. Neuromuscular block during thoracotomy was produced with a single dose of pancuronium 0.1 mg kg 1 i.v. Body temperature was measured with a rectal thermometer and kept within physiological limits by a heating pad and an infrared lamp. After median sternotomy and pericardiotomy, an ultrasonic flow probe was placed around the ascending aorta in order to measure left ventricular stroke Figure 1 Experimental preparation volume minus coronary flow volume (4S or 6S ultrasonic flow probe, T 208, Transonic Systems Inc., Ithaca, NY, USA). Left ventricular (LV) pressure was monitored using a catheter tip manometer (Micro-Tip Pressure Transducer, Sensodyn S PO SF-1, Braun Melsungen AG, Melsungen, Germany) introduced via the left atrium. A ligature snare was passed around a major coronary artery for later occlusion. After completion of the preparation, the thoracotomy was covered with plastic film to lessen evaporative and convective heat loss. Temperature was measured inside the pericardial cradle (GTH 1160, Digital Thermometer, Geisinger Electronic, Germany) and maintained at C by adjusting the heating pad and the infrared lamp. Figure 1 illustrates the preparation of the heart. EXPERIMENTAL PROGRAMME Fifteen minutes after surgery had been completed, baseline measurements were performed. In two rabbits, halothane was given in concentrations of 0.5, 0.75, 1.0 and 1.5 MAC in order to assess the haemodynamic effects of halothane on normal myocardium in this experimental preparation during -chloralose anaesthesia. For the ischaemia reperfusion experiments, after baseline measurements the prepared coronary artery was occluded by tightening the snare. The effectiveness of this manoeuvre was verified by the appearance of epicardial cyanosis and ECG changes. Ventricular fibrillation during coronary occlusion was treated by electrical defibrillation (5 J, DCS261 Defibrillator, Piekser, Ratingen, Germany). After 30 min of occlusion, the snare occluder was released. Reperfusion was verified by the disappearance of epicardial cyanosis. After 120 min of reperfusion, the heart was arrested by injection of 20 ml of potassium chloride solution 16 mmol litre 1 into the left atrium, quickly excised and mounted on a modified Langendorff apparatus for perfusion with ice-cold normal saline via the aortic root at a perfusion pressure of 40 cm H 2 O in order to wash out intravascular blood. After 5 min of perfusion, the coronary artery was reoccluded and the remainder of the myocardium was perfused through the aortic root with 0.2% Evans blue in normal saline for 10 min. Intravascular Evans blue was then washed out by perfusion with normal saline for 5 min. This treatment identifies the area at

3 90 British Journal of Anaesthesia risk as unstained. The heart was then cut into transverse slices, 2 mm thick. The weight of each slice was measured and the slices were stained in buffered 0.75% triphenyltetrazolium chloride solution at 38 C to identify viable and necrotic tissue within the area at risk. 18 The area at risk, and the infarcted area, were determined by planimetry. In the rabbit, the volume of the area at risk has to exceed a certain limit before infarction develops. 19 Therefore, animals with an area at risk of less than 0.5 g were excluded from analysis. Eight rabbits underwent the ischaemia reperfusion programme without further intervention (control group). In seven rabbits, halothane was added to the inspired gas (Vapor 19.3, Drägerwerke AG, Lübeck, Germany) starting 3 min before reperfusion and continued for the first 15 min of the reperfusion period (halothane group). Halothane was titrated to an end-tidal concentration of 1.4% (Datex Capnomac Ultima, Division of Instrumentarium Corp., Helsinki, Finland) which corresponds to 1.0 MAC in rabbits. 20 In seven rabbits, the haemodynamic effects of halothane were antagonized by simultaneous infusion of noradrenaline g kg 1 min 1 (halothane noradrenaline group). DATA ANALYSIS LV pressure, its first derivative dp/dt, AOP and stroke volume (SV) were recorded continuously on an ink-recorder (Recorder 2800, Gould Inc., Cleveland, OH, USA) and stored on a videotape recorder (SL-C 30 PS, Sony, Tokyo, Japan) using pulse code modulation (VPMD 8 12, Fa. Heim, Bergisch Gladbach, Germany) for later playback and analysis. The data were digitized using an analogue to digital converter (Data Translation, Marlboro, MA, USA) at a sampling rate of 500 Hz and processed later on a personal computer. Haemodynamic variables Global systolic function was measured in terms of LV peak systolic pressure (LVPSP) and maximum rate of increase in pressure (dp/dtmax). Global LV end-systole was defined as the point of minimum dp/dt, 21 LV end-diastole as the beginning of the sharp upslope of the LV dp/dt tracing. The time constant of decrease in LV isovolumic pressure ( ) was used as an index of LV diastolic function. 22 was calculated according to the formula: = t Pt P0 e (1) where P t instantaneous pressure; P 0 LV pressure at end-systole and time constant of decrease in isovolumic pressure. Cardiac output (CO) was calculated from stroke volume and heart rate, rate pressure product (RPP) from heart rate and LVPSP, and systemic vascular resistance (SVR) from mean AOP and CO, assuming a right atrial pressure of 0 mm Hg in the openchest preparation. Statistical analysis Data are presented as mean (SEM). The effects of halothane on normal myocardium were assessed by one-way analysis of variance (ANOVA) for repeated observations. In the ischaemia reperfusion experiments, statistical analysis was performed by two-way ANOVA for time and treatment (experimental group) effects. If an overall significance between groups was found, comparison was made for each time using one-wa y ANOVA followed by the Tukey Kramer post-test when appropriate. Results We used 29 animals; one animal died during Table 1 Effect of halothane on normal myocardium. In two rabbits, the effect of increasing doses of halothane was tested during α-chloralose anaesthesia. LVPSP and LVEDP Left ventricular peak systolic and end-diastolic pressure, respectively; dp/dtmax maximum rate of increase in left ventricular pressure; CO cardiac output; SVR systemic vascular resistance; time constant of decrease in isovolumic left ventricular pressure Halothane Baseline 0.5 MAC 0.75 MAC 1.0 MAC 1.5 MAC LVPSP (mm Hg) No No LVEDP (mm Hg) No No dp/dtmax (mm Hg s 1 ) No No CO (ml min 1 ) No No SVR (mm Hg litre 1 min) No No (ms) No No

4 Halothane and reperfusion injury in vivo 91 Figure 2 Analogue registration showing selected times from one experiment of the halothane treated group. LVP Left ventricular pressure, dp/dt rate of change of LVP, AOP aortic pressure, AOF aortic blood flow. induction of anaesthesia, two died from recurrent ventricular fibrillation during coronary occlusion and two animals were excluded because the size of the area at risk was less than 0.5 g. In the remaining 24 animals, complete data sets were obtained (control group, n 8; halothane group, n 7; halothane noradrenaline, n 7; halothane without coronary occlusion, n 2). Four animals had periods of ventricular fibrillation during coronary occlusion; two showed spontaneous defibrillation within 30 s and two needed electrical defibrillation. HAEMODYNAMIC FUNCTION Effects of halothane on normal myocardium In two rabbits, the effect of halothane was tested on normal myocardium (table 1). There was a concentration dependent decrease in LVPSP, dp/dtmax and SVR. CO was maintained at lower concentrations until 1.0 MAC (the concentration used in the ischaemia reperfusion experiments) and decreased at 1.5 MAC. There was prolongation of, reflecting impairment of left ventricular relaxation at higher halothane concentrations. Ischaemia reperfusion experiments Figure 2 shows an analogue reading of the variables in one experiment. Haemodynamic variables are Figure 3 Line plot showing the time course of left ventricular peak systolic pressure (LVPSP), cardiac output (CO) and heart rate (HR) during the study. Data are percentage changes (SEM) from baseline. Mean baseline LVPSP was 96 (SEM 6) mm Hg in the control group (n 8), 91 (8) mm Hg in the halothane group (n 7) and 89 (7) mm Hg in the halothane noradrenaline group (n 7). Baseline CO was 274 (20) ml min 1 in the control group, 309 (30) ml min 1 in the halothane group and 285 (36) ml min 1 in the halothane noradrenaline group. Baseline HR was 227 (8) min 1 in the control group, 226 (11) min 1 in the halothane group and 236 (11) min 1 in the halothane noradrenaline group. Occl. Time of coronary occlusion; drug time of halothane or halothane and noradrenaline administration, respectively. *P 0.05 vs control; P 0.05 halothane vs halothane noradrenaline. P 0.05 vs baseline conditions.

5 92 British Journal of Anaesthesia Table 2 Haemodynamic variables during ischaemia reperfusion expetiments. Data are mean (SEM). dp/dtmax Maximum rate of increase in left ventricular pressure; SVR systemic vascular resistance; RPP rate pressure product; LVEDP left ventricular enddiastolic pressure; = time constant of decrease in isovolumic left ventricular pressure. *P<0.05 compared with control group; P<0.05 compared with baseline conditions; P<0.05 compared with the halothane group Baseline Coronary occlusion Reperfusion 5 min 10 min 25 min 5 min 15 min 30 min 60 min 120 min LVEDP (mm Hg) Control 7.5 (0.8) 145 (11) 138 (12) 143 (10) 147 (15) 149 (10) 136 (10) 137 (13) 100 (19) Halothane 7.9 (1.1) 140 (19) 138 (18) 163 (23) 157 (15) 116 (14) 151 (31) 173 (33) 143 (22) Halothane noradrenaline 7.2 (1.0) 130 (21) 114 (18) 106 (24) 192 (50) 195 (38) 103 (12) 83 (23) 124 (31) dp/dtmax (mm Hg s 1 ) Control 4635 (379) 84 (4) 83 (5) 76 (8) 64 (4) 73 (3) 72 (3) 67 (4) 61 (8) Halothane 4039 (481) 86 (3) 90 (4) 83 (1) 43 (6)* 37 (5)* 60 (7)* 63 (9) 59 (9) Halothane noradrenaline 4149 (577) 82 (6) 81 (7) 85 (9) 51 (4)* 49 (5)* 50 (5)* 50 (6)* 47 (5) SVR (mm Hg litre 1 min) Control 311 (26) 102 (3) 104 (4) 98 (5) 100 (4) 96 (3) 95 (3) 94 (3) 85 (8) Halothane 227 (24) 101 (3) 100 (3) 101 (2) 75 (7)* 62 (7)* 81 (5)* 76 (4)* 71 (6) Halothane noradrenaline 234 (55) 101 (3) 97 (6) 97 (4) 85 (7)* 83 (7)* 65 (5)* 68 (7)* 66 (8) RPP (mm Hg min ) Control 22.2 (1.9) 92 (2) 92 (2) 83 (8) 84 (5) 89 (4) 85 (5) 81 (5) 70 (7) Halothane 20.4 (2.0) 91 (3) 94 (2) 90 (8) 68 (8) 56 (8)* 74 (5) 79 (8) 72 (1) Halothan noradrenaline 21.6 (2.6) 90 (5) 85 (8) 86 (7) 86 (7) 81 (8) 67 (7) 68 (8) 60 (8) (ms) Control 17.1 (2.0) 115 (6) 115 (7) 119 (9) 121 (11) 122 (6) 113 (4) 119 (6) 126 (15) Halothane 17.9 (1.2) 111 (7) 121 (9) 135 (13) 175 (27)* 164 (12)* 142 (7) 132 (5) 134 (10) Halothane noradrenaline 18.8 (1.9) 128 (10) 129 (8) 124 (14) 137 (16) 136 (12) 130 (7) 127 (13) 180 (29) summarized in figure 3 and table 2. During baseline conditions, the groups were comparable in heart rate (control group 227 (8) beat min 1 ; halothane group 226 (11) beat min 1 ; halothane noradrenaline group 236 (11) beat min 1 ; P 0.74), LVPSP (control group 96 (6) mm Hg; halothane group 91 (8) mm Hg; halothane noradrenaline group 89 (7) mm Hg; P 0.76) and CO (control group 274 (20) ml min 1 ; halothane group 309 (30) ml min 1 ; halothane noradrenaline group 285 (36) ml min 1 ; P 0.65). SVR tended to be higher in the control group during baseline conditions (control group 311 (26) mm Hg litre 1 min; halothane group 227 (24) mm Hg litre 1 min; halothane noradrenaline group 234 (55) mm Hg litre 1 min; P 0.18). RPP as a major determinant of myocardial oxygen consumption was similar in all groups (control group 22.2 (1.9) mm Hg min ; halothane group 20.4 (2.0) mm Hg min ; halothane noradrenaline group 21.6 (2.6) mm Hg min ; P 0.31). In all groups, coronary occlusion was accompanied by a small reduction in LVPSP (by 13%), dp/dtmax (by 20%) and CO (by 16%) (table 2, fig. 3). Consequently, SVR remained unchanged. The variables of diastolic function showed prolongation of the isovolumic relaxation phase (increase in by 26%) and an increase in LVEDP by 2.9 mm Hg during coronary occlusion (table 2) (all values at 25 min of coronary occlusion). In the untreated controls, reperfusion led to a small initial recovery in LVPSP (to 90% of baseline) and CO (to 88% of baseline) at 15 min of reperfusion. Halothane 1.0 MAC, given during the first 15 min of reperfusion, led to a further decline in LVPSP to 55% of baseline values and a decrease in CO (to 66%) and SVR (to 62%). After discontinuation of halothane at 15 min of reperfusion, haemodynamic variables recovered to values not different from controls. In the halothane noradrenaline group, the decrease in CO and LVPSP during halothane administration was prevented and values were not different from controls. After discontinuation of both drugs, CO remained unchanged, but SVR, LVPSP and dp/dtmax were reduced at 30 min of reperfusion to 70%, 50% and 69% of baseline values, respectively. Heart rate remained unchanged during the experiment in all groups, and consequently RPP was changed primarily in parallel with changes in LVPSP. At the end of the 120-min reperfusion period, LVPSP and CO were reduced to a similar degree in all three groups compared with baseline. INFARCT SIZE Mean LV weight was 5.55 (0.21) g; there were no differences between groups (data for individual animals are given in table 3). The ischaemic reperfused area (area at risk) constituted 30.4 (2.3) % of the LV. In the control group, infarct size was 49.3 (5.7) % of the area at risk (fig. 4). Infarct size was reduced markedly in halothane treated animals (32.2 (2.9) % of the area at risk, P 0.05 vs control), and to a similar extent in the halothane noradrenaline treated animals (30.0 (3.4) % of the area at risk, P 0.05 vs control). The relationship between the size of the area at risk and amount of infarcted tissue is shown in figure 5. The absolute size of the infarcted tissue correlated closely with the size of the area at risk in all groups (control, r 0.98; halothane, r 0.69; halothane noradrenaline,

6 Halothane and reperfusion injury in vivo 93 Table 3 Left ventricular weight, area at risk and infarct size. Data for individual rabbits Rabbit No. Left ventricular weight (g) Area at risk (% of left ventricular weight) Infarct size (% of area at risk) Control Halothane Halothane noradrenaline Figure 4 Infarct size as a percentage of the area at risk. Open symbols single data points, filled symbols mean (SEM). r 0.81). The slope of the regression line relating infarct size and area at risk size was reduced markedly in the halothane (0.21 (0.10), P 0.001) and halothane noradrenaline groups (0.37 (0.12), P 0.01) compared with the control group (0.86(0.07)). Discussion CRITIQUE OF METHODS Variables that are considered to be important determinants for development of myocardial infarction are duration of ischaemia, collateral blood flow towards the ischaemic area, size of the area at risk and myocardial temperature during ischaemia. The rabbit has a consistently small collateral circulation 23 Figure 5 Scatterplot of the relationship between infarct size and size of the area at risk. Slopes of regression lines were smaller in the halothane (P 0.001) and halothane noradrenaline groups (P 0.01). Control group, y 0.86 (SEM 0.07) x 0.54 (0.13); halothane group, y 0.21 (0.10) x 0.18 (0.19); halothane noradrenaline group, y 0.37 (0.12) x 0.09 (0.17). and therefore it was not necessary to assess collateral blood flow in the ischaemic area. The rabbit heart, however, may have other ways of supplying oxygen to parts of the area at risk, probably by diffusion or through retrograde thebesian vein circulation. 19 Consequently, there is virtually no infarction in very small areas at risk. 19 Therefore, we excluded from the analysis animals with an area at risk of less than 0.5 g. In addition, we analysed the relationship between infarct size and size of the area at risk to avoid distortion of the results by unaccounted differences in size of the area at risk between groups. Temperature was shown recently to affect infarct size even in the normothermic range to a large extent in the open-chest rabbit; a change in infarct size of approximately 10% for each 1 C was reported. 24 Therefore, in this study, temperature was measured inside the pericardium and kept within a narrow range of C. All experiments were carried out during anaesthesia with -chloralose. This type of anaesthesia maintains near normal cardiovascular reflexes comparable with the awake state 25 and is a classical anaesthetic for physiological and pharmacological experiments. 26 An anti-ischaemic action of chloralose has not been found, but we cannot completely exclude interference with reperfusion injury in our study. As the danger of impending hypercontracture in the reoxygenated myocardial cell is evoked by reactivation of mitochondrial energy production, 30 a protective substance should be present at the onset of reperfusion. In anoxic reoxygenated rat cardiomyocytes, we found that intracellular calcium homeostasis recovered within 5 min 10 and after this period administration of halothane could be discontinued without danger of hypercontracture. For these reasons, we administered halothane 3 min before reperfusion in order to have a stable end-tidal concentration corresponding to 1.0 MAC at the onset of reperfusion, and discontinued halothane after 15 min of reperfusion. Because theorabbit has virtually no collateral circulation 23 allowing

7 94 British Journal of Anaesthesia halothane to reach the ischaemic myocardium, and because there was only a short period of administration during the last 3 min of ischaemia, a potential anti-ischaemic effect is unlikely to have contributed substantially to the protection. INTERPRETATION OF RESULTS In addition to the direct influence on the reperfused myocardium, the haemodynamic effects of halothane during the initial 15 min of reperfusion could have influenced the extent of myocardial damage. During this period, halothane reduced LVPSP and RPP, as a variable of myocardial oxygen demand, by 35% and 34%, respectively. These changes in LVPSP were similar to the effects seen in rabbits without ischaemia reperfusion. While CO was nearly maintained at 1.0 MAC of halothane in norma l hea rts, the sa me concentra tion wa s accompanied by a reduction in CO (by 18%) in the presence of regional ischaemia reperfusion. The effects on myocardial reperfusion injury that result from these changes in ventricular loading conditions and the reduction in myocardial inotropy are not known exactly and experimental data are contradictory. In an in vivo dog heart preparation, with 2 h of coronary occlusion the protective effect of 100% ventricular unloading by cardiopulmonary bypass for a 4-h reperfusion period was investigated and resulted in 50% reduction in infarct size. 39 In isolated rabbit hearts, however, complete ventricular unloading had no effect on infarct size. 40 To exclude possible effects of a reduction of ventricular loading conditions by halothane during the initial reperfusion period, we antagonized the negative inotropic and vasodilator effects by continuous infusion of noradrenaline during halothane administration. This treatment effectively prevented the haemodynamic effects during administration of halothane, but there was a small decrease in SVR and, consequently, LVPSP after discontinuation of both drugs. However, these changes were small (LVPSP decreased by 22%) and were too late during reperfusion to explain the marked reduction in infarct size, because lethal reperfusion injury starts with resumption of mitochondrial energy production at the early onset of reperfusion. 30 In addition to its global haemodynamic effects, noradrenaline also acts as a coronary vasoconstrictor. Although coronary flow was not measured in this study, significant coronary vasoconstriction produced by noradrenaline is unlikely because coronary vasoconstriction can be expected to increase infarct size, and in our study the reduction in infarct size by halothane was unchanged by the presence of noradrenaline. In conclusion, it is unlikely that the haemodynamic effects of halothane contributed to the observed myocardial protection. Reperfusion of ischaemic myocardium after temporary coronary occlusion initiates structural and biochemical changes that limit the amount of potentially salvageable myocardium (reperfusion injury). 12 In previous in vitro studies, we found that halothane markedly reduced the oxygen paradox type of reperfusion injury in isolated hearts 8 and isolated cardiomyocytes. 9 The oxygen paradox was described originally for hypoxic-perfused and reoxygenated isolated hearts and is characterized by abrupt development of hypercontracture and cytolysis after re-supply of oxygen. 31 Cellular hypercontracture depends on several factors: re-supply of ATP by reactivation of oxidative phosphorylation and cycles of transient release and reuptake of Ca 2 by the sarcoplasmic reticulum. 32 These calcium cycles trigger uncontrolled activation of the contractile elements, 11 leading finally to cytolysis and cell death, most likely as a result of mutual mechanical disruption of the cells In isolated anoxic reoxygenated cells, halothane prevented these Ca 2 oscillations by an action on the Ca 2 - dependent Ca 2 -release channel of the sarcoplasmic reticulum This study has extended these findings to the situation of reperfusion after regional myocardial ischaemia in vivo, leading to a marked reduction in myocardial infarct size when given during the initial reperfusion period. Other cellular mechanisms affected by halothane are calcium influx via voltage-dependent calcium channels and inhibition of the Na H exchange mechanism. 35 The former is important for the negative inotropic effect of halothane in normal myocardium, 36 but may play only a minor role in the development of lethal reperfusion injury. 37 The latter mechanism has been shown to protect against reperfusion injury 38 by preserving intracellular acidosis and preventing pathological calcium cycling. 11 However, in isolated anoxic reoxygenated cells, 10 recovery of intracellular acidosis was not prolonged in the presence of halothane, suggesting that inhibition of the Na H exchanger by halothane is of minor importance during reperfusion. In vivo, the activated leucocyte is an additional potential cause of (late) lethal reperfusion injury. 41 Although leucocyte activation and accumulation is a consequence of initial state of tissue injury, leucocytes may cause further injury by synthesizing and releasing a variety of mediators, including oxygenderived free radicals and oxidants. 41 By plugging capillaries, they are thought to play an important role in the delayed no-reflow phenomenon, 42 resulting in persistent ischaemia. Consequently, leucocyte depletion during reperfusion reduces the noreflow and infarct zones in dog. 43 Halothane anaesthesia may reduce leucocyte activity in vivo A reduction in post-ischaemic adhesion of (human) leucocytes in the coronary system of guinea pigs was shown recently in the presence of halothane in vitro. 46 Actions on leucocyte activation may have contributed to the myocardial protection observed in this study. Myocardial reperfusion injury may occur in a variety of clinical settings such as thrombolysis, percutaneous balloon angioplasty and after periods of cardiac arrest during heart surgery with cardiopulmonary bypass. Using isolated rat hearts, it was possible to show that administration of halothane during reperfusion conferred additional cardiac protection after cardioplegic arrest. 47 Further studies must show if the cardioprotective effects of halothane against reperfusion injury are clinically significant.

8 Halothane and reperfusion injury in vivo 95 In summary, we found that treatment with 1.0 MAC of halothane during the early reperfusion period after regional myocardial ischaemia reduced markedly the extent of myocardial infarction in vivo. The reduction in infarct size was similar if negative inotropy and vasodilatation were antagonized by infusion of noradrenaline. Therefore, the protection was probably not caused by the haemodynamic effects of halothane, but by a specific action on the reperfused myocardium. Acknowledgement We thank Ms Elke Hauschildt, Mr Thomas Comfère, CAND. MED and Mr Michael González, CAND. MED. References 1. Spieckermann PG, Brückner JB, Kübler W, Lohr B, Bretschneider HJ. Preischemic stress and resuscitation time of the heart. Verhandlungen der Deutschen Gesellschaft für Herz-und Kreislaufforschung 1969; 33: Cope DK, Impa sta to WK, Downey JM. Inha la tiona l anesthesia protects the ischemic myocardium from infarction. 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