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Isoflurane Anesthesia Preserves Liver and Lung Mitochondrial Oxidative Capacity After Gut Ischemia–Reperfusion

Collange, Olivier MD*,†; Charles, Anne-Laure PhD†,‡; Noll, Eric MD*,†; Bouitbir, Jamal PhD†,‡; Zoll, Joffrey PhD†,‡; Piquard, François MD, PhD; Diemunsch, Pierre MD, PhD*,†; Geny, Bernard MD, PhD†,‡

doi: 10.1213/ANE.0b013e3182367a10
Critical Care, Trauma, and Resuscitation: Brief Report
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BACKGROUND: Lung and liver dysfunction is involved in gut ischemia–reperfusion (IR)–induced multiple organ failure. We compared the effects of ketamine and isoflurane on liver and lung mitochondrial oxidative capacity after gut IR.

METHODS: Adult male Wistar rats were randomized into 4 groups (controls and gut IR receiving either intraperitoneal ketamine or inhaled isoflurane). Maximal oxygen consumption and the activity of respiratory chain complexes were measured on isolated liver and lung mitochondria.

RESULTS: Gut IR significantly impaired liver and lung mitochondrial oxidative capacity when using ketamine but not isoflurane.

CONCLUSIONS: Isoflurane preserved liver and lung mitochondrial oxidative capacity after gut IR.

Published ahead of print November 3, 2011 Supplemental Digital Content is available in the text.

From the *Pôle Anesthésie Réanimation Chirurgicale, SAMU, Hôpitaux Universitaires de Strasbourg, Strasbourg, France; Laboratoire EA 3072, Institut de Physiologie, Faculté de Médecine, Université de Strasbourg, Strasbourg, France; Service de Physiologie et d'Explorations Fonctionnelles, Pôle de Pathologie Thoracique, Hôpitaux Universitaires de Strasbourg, Strasbourg, France.

Funding: This work was partially funded by the Cercle des Anesthésites Réanimateurs d'Alsace (CARA) and the Association des Praticiens Hospitaliers d'Anesthésie et de Réanimation (APHAR-SAMU 67), 2 nonprofit organizations.

The authors declare no conflict of interest.

Reprints will not be available from the authors.

Address correspondence to Olivier Collange, MD, Pôle Anesthésie, Réanimation Chirurgicale, SAMU, Hôpitaux Universitaires de Strasbourg, Avenue Molière, 67098 Strasbourg, France. Address e-mail to olivier.collange@chru-strasbourg.fr.

Accepted August 9, 2011

Published ahead of print November 3, 2011

Gut ischemia–reperfusion (IR) is a common occurrence after an ischemic event (acute mesenteric ischemia, aortic occlusion, or gut hypoperfusion associated with shock) followed by restoration of bloodflow. All types of shock cause disproportionate splanchnic hypoperfusion, and with resuscitation, gut reperfusion leads to systemic inflammatory response and remote organ injury.1 In the clinical setting of shock, persistent gut IR episodes are predictive of multiple organ failure and death.2,3

Intestinal venous circulation is coupled in series with the hepatic and pulmonary vascular systems. Both the liver and lung are closely involved in the deleterious effects induced by gut IR, partly because they trap circulating activated leukocytes.4 However, brief exposure to volatile anesthetics, such as isoflurane, can protect against IR-induced injury in many organs, including the liver and lungs.5,6

Mitochondria have a key role in cell function and survival and mitochondrial dysfunction would appear to be involved in sepsis-induced multiple organ failure.7 Previous studies on gut IR have approached mitochondrial function indirectly by measuring serum acetoacetate/3-hydroxybutirate levels as an index of hepatic mitochondrial redox state and pyridine nucleotide (NADH) autofluorescence as an indicator of mitochondrial oxygen consumption and redox status.4,8 Horie et al. have shown that gut IR significantly increases liver NADH by 52%, a sign of liver mitochondria impairment.4

However, the impact of gut IR on both liver and lung mitochondria remains incompletely understood. The objective of our study was to investigate whether gut IR impairs liver and lung mitochondrial function and whether inhaled isoflurane, when compared with intraperitoneal ketamine, can reduce any potential deleterious effects of gut IR on mitochondrial oxidative capacity.

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METHODS

Animals and Experimental Design

Male Wistar rats weighing 350 ± 30 g were randomized into 4 groups of 8 animals (2 control groups [C] and 2 gut-IR groups [IR]) receiving either ketamine or isoflurane.

The animals were placed on heating pads to maintain body temperature at 37°C and kept under spontaneous ventilation during the experiment. Anesthesia was induced and maintained by either intraperitoneal ketamine (induction dose: 20 mg/kg) or inhaled isoflurane (1%–2%) from the beginning to the end of the experiment. Gut IR was induced by 60 minutes of superior mesenteric artery clamping followed by 60 minutes of reperfusion, as previously described.9

Procedures were conducted in accordance with the institutional guidelines for the care and use of laboratory animals, and the study was approved by the institutional animal care committee of the University of Strasbourg (CREMEAS authorization no. AL/03/11/06/09).

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Measurements

All variables were determined at the end of reperfusion (IR) or equivalent time (C).

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Arterial Blood Gases

Arterial blood was sampled (Pulsator® syringe, Portex, Keene, NH) after 1 hour of ischemia and 1 hour of reperfusion and immediately analyzed for arterial partial oxygen (PaO2) and partial carbon dioxide (PaCO2) pressure using an automated blood gas analyzer (Radiometer ABL 725®, Copenhagen, Denmark). Blood gases and pH values were corrected to the rats' rectal temperature measured at the end of the experiment. Throughout the experiment, the rats breathed spontaneously with 1 L/min of oxygen support.

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Arterial Blood Lactate

Arterial blood lactate levels were used as a quantitative marker of gut IR and measured using a micromethod device (Lactate Pro, LT1710, Arkray, KGK, Japan).10

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Mitochondrial Oxidative Capacity

The oxidative capacity of isolated liver and lung mitochondria, which were obtained by differential centrifugation in ice-cold buffer, was assessed by measuring oxygen consumption with a Clark-type electrode (Strathkelvin Instruments, Glasgow, Scotland). Maximal mitochondrial respiration (State 3) rates were measured in the presence of a saturating amount of adenosine diphosphate as a phosphate acceptor.

The relative contributions of respiratory chain complexes I to IV to the global mitochondrial respiratory rates were determined. When State 3 was recorded, electron flow went through complexes I, III, and IV. Complex I was blocked with amytal (0.02 mM). Using succinate 25 mM, we obtained the activities of complexes II, III, and IV (State 3+Suc). The activity of complex IV (cytochrome c oxydase) was then determined by the further addition of N,N,N′,N′-tetramethyl-p-phenylenediamine dihydrochloride (TMPD, 0.5 mM) and ascorbate (0.5 mM) as an artificial electron donor to cytochrome c oxydase (State 3+TMPD).11

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Statistical Analysis

Results were expressed as means ± SD. Two-way analysis of variance was used to compare groups; significance levels were set at P < 0.05. We used GraphPad Prism version 5.03 for Windows (GraphPad Software, San Diego, CA).

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RESULTS

Arterial Blood Gases

During both ketamine and isoflurane anesthesia, gut IR tended to decrease PaO2 values (P = 0.07) and significantly reduced pH and PaCO2 values (P < 0.05) (hyperventilation compensating metabolic acidosis) (Table 1).

Table 1

Table 1

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Blood Lactate

Gut IR led to a similar increase in blood lactate levels whether ketamine (from 1.2 ± 0.5 to 2.9 ± 0.8 mmol/L; +142%; P < 0.001) or isoflurane (from 1.2 ± 0.2 to 2.9 ± 0.5 mmol/L; +142%; P < 0.001) was used to induce and maintain anesthesia.

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Liver and Lung Mitochondrial Oxidative Capacity

There was no significant difference between ketamine and isoflurane control groups.

During ketamine anesthesia, gut IR significantly reduced maximal oxidative capacity (State 3) by 50% in isolated liver mitochondria (from 51 ± 11 to 24 ± 45 μmolO2/min/g; P < 0.05) (Fig. 1A). It also significantly reduced respiratory chain complex activity, by 27% for State3+Suc activity (from 51 ± 8 to 32 ± 13 μmolO2/min/g; P < 0.05) and by 30% for State 3+TMPD activity (from 69 ± 10 to 48 ± 15 μmolO2/min/g; P < 0.05).

Figure 1

Figure 1

In isolated lung mitochondria, maximal oxidative capacity was also reduced (by 30%), but not significantly (from 23 ± 4 to 16 ± 9 μmolO2/min/g; P > 0.05) (Fig. 1B). The decrease in respiratory chain complex activity was, however, significant: activity decreased by 41% for State3+Suc activity (from 27 ± 5 to 16 ± 8 μmolO2/min/g; P < 0.001) and by 17% for State 3+TMPD activity (from 47 ± 8 to 39 ± 13 μmolO2/min/g; P < 0.05).

During isoflurane anesthesia, gut IR did not impair either liver or lung mitochondrial respiration rates (Figs. 1A and 1B).

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DISCUSSION

Isoflurane anesthesia preserved liver and lung mitochondrial respiration in our gut IR model using isolated mitochondria. In this model, IR caused the same gut injury, as assessed by lactate levels, regardless of whether intraperitoneal ketamine or inhaled isoflurane was used to induce and maintain anesthesia. However, when ketamine was used, there was significant, early impairment of liver and lung oxidative capacity, whereas this did not occur with isoflurane. Complex I activity in the lung tended to be less impaired than in the liver, but this tendency was not statistically significant. Further studies should determine the protective mechanism specific to each of these organs.

Our experiment simulated clinical practice when hypovolemia triggers mesenteric hypoperfusion during surgery. Our observations are consistent with an indirect protective effect of isoflurane on electron transport in lung and liver mitochondria that can be attributed to either pre- or postconditioning mechanisms.5,12,13 Isoflurane has been shown to protect against IR injury in multiple organs including the liver and lungs, probably by inhibiting cell death and improving mitochondrial function.5,6,12 This protection has been demonstrated when isoflurane is given before (preconditioning)5,12 or after (postconditioning)13 the ischemic period.

Our direct measurements of mitochondrial respiratory capacity support earlier, indirect findings on mitochondrial function.4,8 They provide further evidence of mitochondrial involvement in isoflurane-mediated protection of the liver and lung. Future research should further study the clinical significance of isoflurane administration in patients with gut IR and establish whether isoflurane protection can enhance the prognosis of these patients.

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DISCLOSURES

Name: Olivier Collange, MD.

Contribution: This author helped design the study, conduct the study, analyze the data, and write the manuscript.

Attestation: Olivier Collange has seen the original study data, reviewed the analysis of the data, approved the final manuscript, and is the author responsible for archiving the study files.

Name: Anne-Laure Charles, PhD.

Contribution: This author helped design the study, conduct the study, and analyze the data.

Attestation: Anne-Laure Charles has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.

Name: Eric Noll, MD.

Contribution: This author helped conduct the study and analyze the data.

Attestation: Eric Noll has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.

Name: Jamal Bouitbir, PhD.

Contribution: This author helped conduct the study.

Attestation: Jamal Bouitbir has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.

Name: Joffrey Zoll, PhD.

Contribution: This author helped design the study, analyze the data, and write the manuscript.

Attestation: Joffrey Zoll has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.

Name: François Piquard, MD, PhD.

Contribution: This author helped design the study and analyze the data.

Attestation: François Piquard has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.

Name: Pierre Diemunsch, MD, PhD.

Contribution: This author helped design the study, analyze the data, and write the manuscript.

Attestation: Pierre Diemunsch has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.

Name: Bernard Geny, MD, PhD.

Contribution: This author helped design the study, analyze the data, and write the manuscript.

Attestation: Bernard Geny has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.

This manuscript was handled by: Michael Murray, MD, PhD.

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ACKNOWLEDGMENTS

We thank Fabienne Goupilleau, Isabelle Bentz, Martine Muckensturm, Anne Schmitt, and Anne-Marie Leonardo for assistance. We thank the Cercle des Anesthésites Réanimateurs d'Alsace (CARA) and the Association des Praticiens Hospitaliers d'Anesthésie et de Réanimation (APHAR-SAMU 67), both nonprofit organizations, for financial support.

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© 2011 International Anesthesia Research Society