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From the *Department of Anesthesiology, Zhongnan Hospital, Wuhan University, Wuhan, Hubei, People’s Republic of China; and Department of Anesthesiology, State Key Laboratory of Biotherapy of Cancer, West China Hospital, Sichuan University, Chengdu, Sichuan, Sichuan, People’s Republic of China.
Address correspondence and reprint requests to Yanlin Wang, MB, Professor and Chairman, Department of Anesthesiology, Zhongnan Hospital of Wuhan University, Wuhan 430071, Hubei, China, or Jin Liu, MD, Professor and Chairman, Department of Anesthesiology, State Key Laboratory of Biotherapy of Cancer, West China Hospital, Sichuan University, Chengdu, Sichuan 610041, Sichuan, China. Address e-mail to wangyanlin0510{at}yahoo.com or wuliujin{at}china.com.
Abstract
BACKGROUND: In this study, we examined the cardioprotective effects of parental emulsified isoflurane compared with inhaled isoflurane.
METHODS: Thirty-two rabbits were subjected to 30 min of myocardial ischemia induced by temporary ligation of the left anterior descending coronary artery followed by 3 h of reperfusion. Before left anterior descending coronary artery occlusion, the rabbits were randomly allocated into one of four groups (eight for each group): group C, no ischemia preconditioning treatment; group IS, inhaled isoflurane 1.1% end-tidal; group EI, a continuous infusion of 8% emulsified isoflurane to an end-tidal concentration of 0.64%; and group IN, a continuous infusion of 30% Intralipid® started 30 min. Treatments were started 30 min before ischemia followed by a 15 min washout period for isoflurane groups. Myocardial infarct volume, lactate dehydrogenase, and creatine kinase levels were measured and changes in mitochondrial ultrastructure assessed after 3 h myocardial reperfusion.
RESULTS: Myocardial infarct size 3 h after reperfusion was lower in groups IS and EI compared with groups C and IN (20% ± 8%, 18% ± 8%, 39% ± 6%, and 34% ± 9%, respectively, P < 0.01). There were no differences in myocardial infarct size between groups IS and EI or between groups C and IN. Plasma lactate dehydrogenase and creatine kinase levels were lower in group IS (456 ± 58 U/L and 1725 ± 230 U/L) and group EI (451 ± 54 U/L and1686 ± 444 U/L) 3 h after myocardial reperfusion compared with groups C (676 ± 82 U/L and 2373 ± 529 U/L; P < 0.01). Mitochondrial ultrastructure changes were less pronounced in groups IS and EI compared with group C.
CONCLUSIONS: Our results indicate that, in rabbits, IV emulsified isoflurane provides similar myocardial protection against ischemia-reperfusion injury as inhaled isoflurane.
Ischemic preconditioning is a well known phenomenon in which brief episodes of sublethal ischemia induces robust protection against the deleterious effects of subsequent, prolonged ischemia in a variety of organ systems, including the brain, heart, liver, intestine, kidney, and lung.1 In the heart, inhaled isoflurane mimics ischemic preconditioning, limiting myocardial infarct size to a similar extent.2–10 Emulsified isoflurane is a new lipid-based formulation of the volatile anesthetic that permits IV administration. Prior investigations have demonstrated the feasibility and safety of induction of anesthesia with IV emulsified isoflurane.11–14 Such a preparation may be clinically useful for providing anesthetic preconditioning to organs at risk for ischemic injury in clinical areas outside of the operating rooms, such as the cardiac catheterization laboratory. Therefore, the purpose of this study was to test the hypothesis that IV administration of emulsified isoflurane before ischemia limits myocardial injury to a similar extent as inhaled isoflurane.
METHODS
All experimental procedures used in this investigation were approved by the Animal Experiment Committee of Wuhan University, China, and conformed to the Guide for the Institutional Animal Care and Use Committee of Wuhan University. Furthermore, all procedures were in accordance to the Guiding Principles in the Care and Use of Animals of the American Physiologic Society and the Guide for the Care and Use of Laboratory Animals. Male adult New Zealand white rabbits weighing 2.0–2.5 kg were housed in a room with controlled temperature (24°C ± 1°C) and humidity (55% ± 5%) under 12 h light–dark cycle. They were allowed free access to food and water.
Materials
Intralipid®, 30%, was provided by Huarui Pharmacy, Ltd. (Chengdu, China). Isoflurane was purchased from Abbott Laboratories (Queenborough, Kent, United Kingdom). Emulsified isoflurane, 8%, was provided by the Laboratory of Anesthesiology and Critical Care Medicine, West China Hospital, Sichuan University (Chengdu, China). Emulsified isoflurane was prepared according to the procedures described by Yang et al.13 Briefly, 1.6 mL liquid isoflurane and 18.4 mL 30% Intralipid were mixed in a 20-mL glass ampoule, and the ampoule was sealed using an alcohol blowtorch. The ampoule was then vigorously shaken on a vibrator for 15 min to solubilize isoflurane into the lipid emulsion. The emulsified isoflurane ampoule was opened just before use and the residual drug was discarded. Before this experiment, the stability of 8% emulsified isoflurane was investigated by gas chromatography. There were no changes in isoflurane concentration and no lipid droplets were found during 6 mo of storage at room temperature.
Groups and Experimental Protocols
The rabbits were anesthetized with IV sodium pentobarbiturate (30 mg/kg) via an ear vein; anesthesia was maintained with an IV infusion of pentobarbiturate at a rate of 5 mg · kg–1 · h–1. Body temperature was maintained at 38.5°C during the procedure with a thermostatically controlled heating blanket. Electrocardiograph leads were placed on the limbs. After a midline cervical incision, a tracheotomy was performed and the animal’s lungs were mechanically ventilated using an anesthesia apparatus (Excel 210; Datex-Ohmeda, Madison, WI) with 100% oxygen, a tidal volume of 15 mL/kg, and a respiratory rate of 35 per min adjusted to keep the end-tidal CO2 between 30 and 35 mm Hg (monitored with use of a M1026A monitor; Philips, Suzhou, China). The right femoral artery was cannulated for sampling of blood and for measurement of mean arterial blood pressure (MAP). Heart rate (HR) was determined from the arterial pressure curve. The pericardium was incised and the heart was exposed. A 2.0 silk suture was passed around the proximal part of the left anterior descending coronary artery (LAD) and the ends of the silk suture were threaded through a small vinyl tube to form a snare that could be tightened to occlude, and loosened to reperfuse, the artery. Myocardial ischemia was confirmed by ST-segment elevation on the electrocardiogram and regional cyanosis of the myocardial surface. Reperfusion was confirmed by myocardial blush over the area-at-risk (AAR) when the snare was released.
After 30 min of stabilization, baseline MAP and HR were recorded. The rabbits then underwent a 30-min LAD occlusion, followed by 3 h of reperfusion. Before LAD occlusion, the rabbits were randomly allocated into four groups according to a computer-generated number schedule (eight for each group). The control group (group C) did not receive any preconditioning treatment. The inhaled isoflurane group (group IS) received inhaled isoflurane to an end tidal concentration of 1.1% (corresponding to 0.5 MAC15) for 30 min, followed by a 15-min washout period. In the emulsified isoflurane group (group EI), 4–6 mL of 8% emulsified isoflurane was administered IV at the rate of 1 mL/s and then maintained at an end-tidal concentration 0.64% (corresponding to 0.5 MAC 8% emulsified isoflurane in rabbit,13 unpublished data from our laboratory Xiao-Lin Yang et al. June 2004) for 30 min by adjusting the rate of the microinfusion pump (approximately 4–6 mL · kg–1 · h–1, Silugao High Technology Development CO, Ltd., Beijing, China). The emulsified isoflurane infusion was followed by a 15-min washout period. The Intralipid group (group IN) received 30% Intralipid IV 5 mL bolus and then 5 mL · kg–1 · h–1 for 30 min before ischemia (Fig. 1).
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Measurements
Arterial blood (2 mL) was withdrawn from the femoral artery catheter before ischemia and at the end of the 3 h reperfusion period. The blood was immediately transferred to polyethylene tubes containing 50 µL heparin (250 IU). The blood was then centrifuged at 3000 rpm for 15 min. The plasma was collected and aliquots were stored at –20°C for the determination of lactate dehydrogenase (LDH) activity and creatine kinase (CK) activity in blocks. The LDH and CK activity were assayed by colorimetry with an automatic clinical analyzer (AZROSET, Abbott, US) using commercially available assay kits (Roche CO, Ltd., Basel, Switzerland). Intra- and interassay variations were <5% and 10%, respectively. Detection limits for the LDH and CK kits used are 10 and 15 U/L, respectively. The results were expressed as total LDH and CK release from the heart after the indicated time periods.
Measurement of Myocardial Infarct Size
Myocardial infarct size was measured by the method of Tsai et al.16 by an investigator blinded to experimental group assignment. Briefly, after 3 h of reperfusion, the LAD was reoccluded and 2.0 mL of Uniperse blue dye was injected IV to delineate the AAR. With this technique, the previously nonischemic area appears blue whereas the AAR remains unstained. The AAR was cut out, weighed, and expressed as percentage of total ventricular weight. Ventricular tissue was sliced into sections for incubation with tetrazolium dye (2,3,5-triphenyltetrazolium chloride 1%, Sigma Chemicals, St. Louis, MO) at 37°C in the dark for 30 min to differentiate infarcted (pale) from viable (red) myocardial area. The cardiac apex [0.1 g, pale left ventricular (LV) tissue samples] was fixed for transmission electron microscopy (TEM) with fixative solution (2.5% glutaraldehyde, in 0.1 M phosphate buffer). Tissue samples were "post-fixed" in buffered 1% OsO4, dehydrated a series of ethanols (50%, 70%, 80%, 95%, 100%, and 100%) and embedded in resin. Finally, ultrathin sections (50–70 nm) of myocardium were stained with 1% uranyl acetate and examined under an H-600 TEM (Hitachi, Japan) by an investigator blinded to the assignment of the sections. Rabbits that developed intractable ventricular fibrillation and those with an AAR <15% of total LV mass were excluded from subsequent analysis.
Statistical Analysis
Data analysis among groups was performed using multiple analysis of variance for repeated measures with post hoc analysis by the Student Newman-Keuls q test with Bonferroni’s correction for multiple pairwise comparisons. Differences were considered significant when P was <0.05. All data are expressed as mean ± sd of mean (sd).
RESULTS
Thirty-seven rabbits were instrumented to obtain 32 successful experiments. Five rabbits were excluded because of technical problems during instrumentation or because of the development of intractable ventricular fibrillation (two from group C, one each from groups IS, EI, and IN).
Systemic hemodynamic changes in the four groups of rabbits during the observation period are presented in Table 1. There were no significant differences in hemodynamic measurements among groups at baseline or during the experiment. There were no differences in MAP and HR within each group during the experimental periods compared with baseline.
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Myocardial Infarct Size
Myocardial infarct size expressed as a percentage of the LV AAR for the experimental groups is listed in Table 2. The mean LV dry weight was not different among groups. There was no difference in myocardial infarction size between group C and group IN (39% ± 6% vs 34% ± 9%, P > 0.05). In contrast, myocardial infarct size in group IS and group EI were smaller compared with both groups C and IN (20% ± 8% and 18% ± 8%, respectively, P < 0.01). There were no significant differences in myocardial infarction size between groups IS and EI (P > 0.05).
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TEM
Representative TEM images of LV tissue obtained after 30 min of LAD occlusion and 3 h of reperfusion for the four experimental groups are shown in Figure 2. TEM from a rabbit heart from group C revealed evidence of widespread mitochondrial damage (Fig. 2A), consisting of severe disturbance in the mitochondrial crista arrangement, the loss of mitochondrial matrix substance, the presence of intramitochondrial vacuoles, and areas of disruption of the mitochondrial membrane. Ultrastructural changes observed in LV tissue obtained from group IN were similar to that from group C (Fig. 2D). In contrast, pretreatment with inhaled isoflurane and emulsified isoflurane attenuated mitochondrial injury (Figs. 2, B and C) as demonstrated by intact sarcomeres and less swelling and disruption of the mitochondrium.
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Cardiac Enzymes
Plasma LDH and CK results are shown in Figures 3 and 4. There were no differences in baseline LDH or CK levels among groups. In group C, plasma LDH levels after myocardial ischemia and reperfusion were higher compared with group IS and group EI. Plasma LDH levels in group IN were no different than group C but higher compared with group IS and group EI. Plasma CK levels in group IS and group EI were lower than both group C and group IN (P < 0.01). There were no differences in plasma LDH and CK levels between groups C and IN and between groups IS and EI.
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Previous reports of the IV use of volatile anesthetics, either accidentally in humans or in experimental animals, resulted in death or severe morbidity.17–19 These findings may have resulted from overdosing of the anesthetics and/or direct toxic damage. IV administration of volatile anesthetics may have advantages over the inhaled mode of delivery, such as more rapid induction, less atmospheric pollution, and ease of administration. There have been several investigations reporting the safety and efficacy of IV volatile anesthetics emulsified in lipid in animals, including our own experience using emulsified isoflurane (8% vol/vol).11–14
Anesthetic preconditioning is a phenomenon by which brief exposure to a volatile anesthetic leads to subsequent protection of the myocardium from ischemia-reperfusion injury. Inhaled isoflurane myocardial preconditioning has been demonstrated in several in vitro and in vivo models and in humans with coronary artery disease undergoing coronary artery bypass graft surgery.2–10 Anesthetic preconditioning of the myocardium with isoflurane occurs via multiple mechanisms including activation of intracellular signaling pathways, modulation of extracellular signal-related kinases, and via opening of mitochondrial adenosine triphosphate-dependent potassium channels.3–10 Our study, thus, confirms others showing that inhaled isoflurane (0.5 MAC), given for 30 min before myocardial ischemia, limits the extent of myocardial injury after subsequent reperfusion. This is documented by smaller myocardial infarct volume and smaller release of cardiac enzymes compared with control animals.
The previous measured MAC of emulsified isoflurane (1.28%) from our laboratory in rabbits is less than the MAC for inhaled isoflurane (2.20%). Musser et al.20 and Yang et al.13 demonstrated that MAC of IV emulsified halothane and isoflurane (0.78% in pigs and 1.12% in dogs, respectively) were smaller than the MACs for each anesthetic when given by the inhaled route (1.13% and 1.38%, respectively). Although MAC of emulsified isoflurane and inhaled isoflurane were different, the arterial partial pressures of the anesthetics at MAC were similar, indicating that the partial pressures for isoflurane in the central nervous system at the same anesthetic depth are required regardless of route of administration.13 Yang et al. have further demonstrated that the washout times to reduce the arterial blood concentration by 99% for emulsified isoflurane and inhaled isoflurane in dogs are 209.8 ± 95.6 s and 187.3 ± 49.8 s, respectively.13 These latter results suggest that the 15-min washout period used in this experiment before myocardial ischemia should ensure that minimal isoflurane from either route of administration was present in the blood perfusing the animal hearts.
Chiari et al. found that IV administration of 6.9% emulsified isoflurane at a constant rate (3.5 mL · kg–1 · h–1) for 30 min produced acute preconditioning against myocardial infarction in rabbits.21 Our study extends these findings by showing that the extent of myocardial protection from ischemia/reperfusion afforded by IV emulsified isoflurane is similar to that achieved with inhaled isoflurane when given at the same depth of anesthesia. We further found that a relatively small concentration of IV emulsified isoflurane (0.5 MAC) provided cardioprotection without altering systemic hemodynamics in rabbits. Previous investigations have found that myocardial preconditioning with inhaled isoflurane was dose-dependent and occurred at end-tidal concentrations of inhaled isoflurane from 0.25 MAC to 2 MAC in dogs.22 In humans, anesthetic myocardial preconditioning with inhaled sevoflurane occurred at low concentrations (0.5 MAC).23 The dose–response for myocardial protection with IV emulsified isoflurane remains to be determined.
In our experiment, the rabbits in all groups were anesthetized with pentobarbital. Zaugg et al. reported that pentobarbital had no effect on baseline mitochondrial KATP channel activity, one of multiple signaling pathways for anesthetic and ischemic preconditioning.24 Walsh et al. further found that limitation of myocardial infarct size from ischemic preconditioning was not affected by pentobarbital.25 It is, thus, unlikely that pentobarbital influenced the observed myocardial protection from ischemia/ reperfusion injury in the current study. Finally, another limitation of this study is that blood concentrations of isoflurane were not measured. We cannot, therefore, ensure that similar concentrations of isoflurane were indeed delivered to the animals before myocardial ischemia.
In summary, IV 8% emulsified isoflurane appears to provide anesthetic myocardial preconditioning in rabbits subjected to coronary artery occlusion and reperfusion to a similar extent as that provided by inhaled isoflurane.
Footnotes
Accepted for publication December 11, 2007.
Supported by a grant from the National Research Foundation of Nature Sciences China (No. 30271259) and a grant from 973 program (No. 2005CB522601), Beijing, People’s Republic of China.
REFERENCES
rter L, Keel M, Zaugg M. Sevoflurane inhalation at sedative concentrations provides endothelial protection against ischemia-reperfusion injury in humans. Anesthesiology 2007;106:262–8[Web of Science][Medline]This article has been cited by other articles:
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  E. Lucchinetti, M. C. Schaub, and M. Zaugg Emulsified Intravenous Versus Evaporated Inhaled Isoflurane for Heart Protection: Old Wine in a New Bottle or True Innovation? Anesth. Analg., May 1, 2008; 106(5): 1346 - 1349. [Full Text] [PDF]
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