Department of Anaesthesiology and General Intensive Care Unit, Keio University School of Medicine, Tokyo 160-8582, Japan anesmrsk@sc.itc.keio.ac.jp
Accepted for publication: July 23, 2002
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Abstract |
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Methods. With institutional approval, 91 rats were randomly allocated into one of either caecal-ligation and perforation (CLP: n=50) or sham (Sham: n=41) procedure groups 24 h before the study. After determination of baseline measurements, including cardiac output (CO), myocardial oxygen consumption (mV·O2) and cardiac efficiency (CE; COxpeak systolic pressure/mV·O2), each isolated heart was perfused with or without 2% sevoflurane for 15 min before global ischaemia (pre-ischaemia). After 15 min ischaemia and 30 min reperfusion, all hearts were assessed for functional recovery of myocardium (post-reperfusion).
Results. During the pre-ischaemia period, 2% sevoflurane caused a significant reduction of CO in the CLP group compared with the Sham group. During the post-reperfusion period, both CO (16.9 vs 11.0 ml min1) and CE (11.2 vs 7.7 mm Hg ml1 (µl O2)1) was higher in the sevoflurane-treated vs -untreated hearts from CLP rats, and was accompanied by lower incidence of reperfusion arrhythmia compared with control hearts (8 vs 32%). In contrast, 2% sevoflurane did not provide cardioprotective effects in normal rats.
Conclusions. The current study demonstrates that pre-treatment with sevoflurane minimizes myocardial dysfunction and the incidence of reperfusion arrhythmia after brief ischaemic insults in septic hearts.
Br J Anaesth 2002; 89: 896903
Keywords: anaesthetics volatile, sevoflurane; heart, reperfusion arrhythmia; infection, sepsis
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Introduction |
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Sepsis, characterized by a marked elevation of tissue oxygen demand, is frequently accompanied by multiple organ failure including myocardial dysfunction even at the early stage of the hyperdynamic circulatory state.10 11 A previous study showed that the ultra-structural architecture of the myocardium, including mitochondria and myofibrils, were swollen and damaged in a large animal model of sepsis.12 Thus, septic myocardium may not be able to utilize oxygen efficiently even under the conditions of supranormal oxygen supply to the myocardium.11 In addition, another study demonstrated that the flow reserve of the coronary circulation to maximize blood flow and oxygen extraction was limited.13 Thus, septic myocardium appears to be more vulnerable to even brief periods of ischaemia and subsequent reperfusion. We, therefore, tested the hypothesis that sevoflurane protected the septic heart from ischaemia to produce a stunned myocardium. In the present study, we used the caecal-ligation and perforation (CLP) model to develop the early stage of sepsis as described previously.14 Using an isolated working heart from CLP-treated rats, we examined whether exposure of 2% sevoflurane served to minimize myocardial dysfunction and the incidence of reperfusion arrhythmia following brief period of ischaemia.
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Materials and methods |
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Animal preparation
Ninety-one male Wistar rats, weighing 310380 g, were studied after a 3- to 7-day period of acclimatization in our laboratory. Water and laboratory chow were available ad libitum after intraperitoneal (i.p.) injection of pentobarbital (30 mg kg1) and the femoral vein was cannulated with a catheter (PE50; Intermedic, Sparks, MD, USA) under sterile conditions. The animals were then randomized into either sham-treatment (Sham) or CLP group. In the latter animals, a laparotomy was performed and a ligature was placed around the caecum immediately distal to the ileocaecal valve. The caecum was then punctured twice with an 18-gauge needle. Following this preparatory surgery, normal saline was infused at 3 ml h1 via the catheter for the next 24 h. This preparation was demonstrated to produce normotensive, hyperdynamic sepsis.14 As the laparotomy per se caused septic responses in rats, the Sham animals in this experiment did not receive open laparotomy as described previously.15
Study procedure
Figure 1 depicts the study procedure. Twenty-four hours after randomization to either the Sham (n=41) or CLP (n=50) group, animals were anaesthetized with sevoflurane in oxygen. After the intravenous (i.v.) injection of heparin (300 IU kg1), the abdomen was opened and 1 ml of blood was taken from descending aorta for subsequent analyses of arterial lactate. Thereafter, the hearts were rapidly excised, mounted on a non-recirculating Langendorff apparatus to allow further preparation, and perfused with modified KrebsHenseleit solution at 37°C (composition in mM: NaCl 120, KCl 4.8, KH2PO4 1.2, MgSO4 1.2, CaCl2 1.25, NaHCO3 25, and glucose 11) as described previously.5 The perfusion buffer was equilibrated with a 95% oxygen/5% carbon dioxide gas mixture, resulting in a buffer PO2 above 60 kPa.
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After 30 min of initial perfusion in working mode, baseline measurements were used. The hearts were further randomized into two subgroups: control or sevoflurane group. The latter subgroup (Sham/SEVO, n=20 or CLP/SEVO, n=25) was perfused for 15 min with modified KrebsHenseleit solution, in which sevoflurane was administered at 2.0% via a calibrated sevoflurane vaporizer (Datex-Ohmeda, Helsinki, Finland). Control hearts were subjected to the same perfusion procedure without administration of sevoflurane (Sham/CONT, n=21 or CLP/CONT, n=25). All isolated perfused hearts underwent 15 min global ischaemia at room temperature (approximately 25°C). The hearts were then re-perfused for 5 min in a Langendorffs perfusion mode, followed by working heart mode at 37°C for 30 min without sevoflurane. As depicted in Figure 1, measurements described below were repeated during the pre-ischaemic and post-reperfusion periods. Hearts were immediately freeze-clamped between blocks of metal cooled to the temperature of liquid nitrogen and stored at 40°C until extraction for high-energy phosphate analyses.
Specific measurements and calculations
Cardiac output (CO) was defined as the sum of coronary and aortic flows in this experimental setting as described previously.16 Left-ventricular pressure (LVP) was continuously monitored with a transducer (post-craniotomy subdural pressure monitoring kit; 110-4G, Neuro Care Group Camino, CA, USA and Life Scope II, Nihon Koden, Tokyo, Japan) and recorded in a digital recorder (PC208, Sony, Tokyo, Japan). Using computer software (Acqknowledge v3.0 data acquisition systems, Biopac Systems, Goleta, CA, USA and Microsoft Excel, Microsoft, WA, USA), LV peak systolic pressure (LVPSP), LV end-diastolic pressure (LVEDP), maximum rate of LV pressure rise (dP/dtmax), and heart rate (HR) were calculated, based on the averages of five beats. LV developed pressure (LVDP), which was regarded as a marker of contractility of the isolated rat heart, was calculated by subtracting LVEDP from LVPSP.17 Reperfusion arrhythmia was defined as the irregular intervals of peak systolic pressure on the pressure monitor 5 min after the initiation of working mode.
PO2 of the perfusion medium obtained at pre-perfusion (in-flow) and right ventricular outflow (out-flow) was determined using a standard blood gas analyser (Chiron 860 series, Chiron Diagnostics Corp., East Walpole, MA, USA). Oxygen delivery was calculated as inflow oxygen tension, in millimetres of mercury, multiplied by oxygen solubility (24 µl ml1 KrebsRingers solution at 760 mm Hg oxygen and 37°C) and coronary flow (ml min1). Myocardial oxygen consumption (mV·O2) was calculated as oxygen solubility multiplied by the difference between in-flow and out-flow oxygen tension and coronary flow. CE was determined as the ratio of cardiac work, the product of CO (ml min1)xLVPSP (mm Hg), to mV·O2 uncorrected for heart weight as described previously.18 19
Biochemical analyses
High-energy phosphates in the myocardium were measured as described previously.20 Briefly, freeze-clamped left ventricular tissue from CLP and Sham animals were first broken in a frozen sample crusher (TK-CM20S, Yaghi Grassware, Tokyo, Japan) and then homogenized with a teflon-coated homogenizer (Iuchi, Tokyo, Japan) in ice-chilled 0.6 N perchloric acid. The samples were centrifuged (3000 g for 10 min), neutralized with 1 N potassium hydroxide, and re-centrifuged. Myocardial creatine phosphate (CrP), adenosine monophosphate (AMP), adenosine diphosphate (ADP), and adenosine triphosphate (ATP) contents were measured using high-performance liquid chromatography with a reverse-phase column (Shim-Pack VP-ODS, Shimazu, Tokyo, Japan). Elution was performed at 1.0 ml min1, and an ultraviolet/visible spectrophotometer (Shimazu, SPD-10A, Tokyo, Japan) at 210 nm was used to detect these phosphates. Tissue contents of these metabolites were reported in micromoles per gram wet tissue.
Statistical analysis
This experiment utilized a three-factor design with a full 2x2 factorial allotment to (CLP/Sham)x(CONT/SEVO). The third factor, ischaemia-reperfusion, was implemented as a repeated measure. Values are described as mean (SD) unless otherwise specified. To compare and contrast the effects of sepsis across the sevoflurane treatment groups before the induction of ischaemia-reperfusion, two-way analysis of variance was employed using a statistical package of SPSS/9.0J for Windows (SPSS Inc., Chicago, IL, USA). After induction of ischaemia-reperfusion, results between baseline and post-reperfusion periods were analysed using repeated-measures analysis of variance. Where appropriate, directed pair-wise comparisons of individual groups were conducted using a Bonferroni-corrected 95% confidence interval. The incidence of arrhythmia was examined using KruskalWallis test. A P value <0.05 was regarded as significant.
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Results |
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Discussion |
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Whilst previous studies showed that exposure to volatile anaesthetics protected healthy myocardium from prolonged irreversible ischaemia by assessing the reduction of infarct size, there were conflicting results as to whether similar profiles could be given to reversible myocardial injury, that is stunned myocardium.7 21 Indeed, sevoflurane treatment in this experimental condition did not show any cardioprotective properties against ischaemia-reperfusion injury in healthy hearts. There are several possibilities to account for such a discrepancy between the present and previous studies. First, sevoflurane concentration might not be high enough to obtain its preconditioning-like effects in healthy hearts. Previous studies demonstrated that the minimum alveolar concentration (MAC) of sevoflurane was significantly reduced from 2.4 to 1.35% level in a porcine model of sepsis.22 Another study using the same CLP-rat model also showed that the MAC required for isoflurane was lowered to a similar extent.23 Given these findings, it is not unreasonable to consider that the potency of anaesthetics on the isolated myocardium is different between the Sham and CLP groups. If equipotent sevoflurane doses were given to each group, this might have protected the healthy heart from ischaemiareperfusion injury to cause stunned myocardium. Indeed, it should be noted that there are conflicting results, possibly caused by different concentrations of anaesthetic agents exposed at different time periods among previous studies.5 9 24 25 Secondly, sevoflurane anaesthesia for the excision of hearts might have already provided preconditioning-like effects even in the control hearts. Although sevoflurane is much less likely to remain in situ after reperfusion compared with other anaesthetics because of its lower bloodgas solubility,26 this possibility could not be fully excluded. On the other hand, the study design to terminate sevoflurane treatment during the reperfusion period could have modified its cardioprotective effects in healthy hearts. Thirdly, ischaemic insults to evoke a stunned myocardium in the present study might not be severe enough to show distinct protective effects of sevoflurane in health where myocardial reserve is more sufficient than in sepsis.11 In other words, if a longer period of ischaemia was applied to this model, protection might have been unmasked. Furthermore, hypothermic global ischaemia as we applied could be another factor to reduce the severity of the ischaemiareperfusion injury compared with normothermic ischaemia as described elsewhere.16 17
Despite the instability of this septic challenge, the CLP model with fluid resuscitation has been considered most relevant to the early stage of sepsis, concomitantly obviating confounding factors like hypotension.12 14 27 In accordance with these findings, we found no significant elevation of arterial lactate 24 h after CLP, indicating that tissue hypoxia was not evident in this model of sepsis. Even under such conditions, both myocardial contractility, expressed as LVDP, dP/dtmax and CO, and heart rate in the CLP group were significantly lower compared with the Sham group at baseline (Table 1), suggesting that not only inotropic but also chronotropic properties synergistically serve in the development of myocardial dysfunction in sepsis. However, it should also be noted that CO was significantly depressed in the early stage of sepsis under a constant afterload in this isolated preparation. Furthermore, the present study shows that mV·O2 is significantly depressed ex vivo with the reduction in coronary flows and CO (Table 1) whereas it is unknown whether mV·O2 in the hyperdynamic state of sepsis is indeed augmented or not. A previous study demonstrated that pro-inflammatory cytokines, released in sepsis, depressed myocardial function accompanied by reduction of mV·O2 through nitric oxide-dependent mechanisms.18 Further investigations are required to clarify this issue.
Neither CLP nor sevoflurane exposure appears to modify myocardial energy levels, indicating that improved high-energy phosphate contents were not singularly important determinants of post-ischaemic ventricular function. Support for this contention was provided by previous studies, which demonstrated the dissociation between ventricular function and ATP levels in post-ischaemic hearts.28 29 Some investigations showed that myocardial high-energy phosphates content correlated neither with myocardial dysfunction in sepsis nor with cardioprotection by ischaemic preconditioning.30 31 Collectively, one possibility could be raised that the significant reduction in CO, accompanied by depressed mV·O2, resulted in stored high-energy phosphates in the CLP group more than those in the Sham group, subsequently preserving the amount of ATP contents in the CLP group. Furthermore, both a reduction of cardiac efficiency and no changes of myocardial high-energy phosphates in the CLP group, indicates a possibility that sepsis reduces the ability of the myocardium to utilize high-energy phosphates for contractile work, but not inhibit mitochondrial respiration. Nevertheless, the current results provide evidence that energy utilization in the septic heart is augmented with treatment of sevoflurane before ischaemia.
There are several limitations to this study. First, despite a physiologically correct direction of perfusion, the isolated working model limits the evaluation of data regarding myocardial function in sepsis. For example, humoral factors like cytokines and circulating inflammatory cells such as leukocytes or platelets, both of which play consequential roles in the development of sepsis, are excluded from the perfusion medium in this study design. Furthermore, because of constant afterload and depressed contractility, the augmentation of CO observed in this early stage of sepsis was not found during the baseline period. Secondly, it could be argued that myocardial function could be evaluated more accurately if the heart rates were kept constant. Contrary to Langendorff mode, however, sufficient heart rate could be obtained in an isolated working heart because of perfusion to left atrium as described previously.19 In addition, under pacing conditions this model could lose the effects of sevoflurane on heart rates, subsequently modifying CO. Finally, the isolation procedure per se may be able to ischaemic-precondition the heart, possibly modifying the outcome. However, as we performed the study in the same manner by a single investigator in random, we believe that the data were comparable at least between the groups.
In conclusion, the current study shows that sevoflurane provides protective effects, in septic but not healthy hearts, against brief ischaemic insults through the improvement of myocardial oxygen utilization. Further investigation is warranted to elucidate whether other anaesthetic preconditioning protects the myocardium against brief or prolonged period of ischaemia in diseased hosts.
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Acknowledgements |
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