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CARDIOVASCULAR ANESTHESIA

Isoflurane Administration Before Ischemia and During Reperfusion Attenuates Ischemia/Reperfusion-Induced Injury of Isolated Rabbit Lungs

Renyu Liu, MD^*, Yuichi Ishibe, MD, PhD^*, Mayumi Ueda, MD^*, and Yannan Hang, MD

*Department of Anesthesiology and Reanimatology, Tottori University Faculty of Medicine, Yonago, Japan; and Department of Anesthesiology, Renji Hospital, Shanghai Second Medical University, Shanghai, China

Address correspondence and reprint requests to Yuichi Ishibe, MD, Department of Anesthesiology and Reanimatology, Tottori University Faculty of Medicine, 36-1 Nishi-cho, Yonago, Tottori 683-8504, Japan. Address e-mail to ishibe{at}grape.med.tottori-u.ac.jp

	Abstract

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To investigate the effects of isoflurane on ischemia/reperfusion (IR)-induced lung injury, we administered isoflurane before ischemia or during reperfusion. Isolated rabbit lungs were divided into the following groups: control (

n = 6), perfused and ventilated for 120 min without ischemia; ISO-control (n = 6), 1 minimum alveolar anesthetic concentration (MAC) isoflurane was administered for 30 min before 120 min continuous perfusion; IR (n = 6), ischemia for 60 min, followed by 60 min reperfusion; IR-ISO1 and IR-ISO2, ischemia followed by reperfusion and 1 MAC (n = 6) or 2 MAC (n = 6) isoflurane for 60 min; ISO-IR (n = 6), 1 MAC isoflurane was administered for 30 min before ischemia, followed by IR. During these maneuvers, we measured total pulmonary vascular resistance (Rt), coefficient of filtration (Kfc), and lung wet to dry ratio (W/D). The results indicated that administration of isoflurane during reperfusion inhibited an IR-induced increase in Kfc and W/D ratio. Furthermore, isoflurane at 2 MAC, but not 1 MAC, significantly inhibited an IR-induced increase in Rt. The administration of isoflurane before ischemia significantly attenuated the increase in IR-induced Kfc, W/D, and Rt.

Implications: Our results suggest that the administration of isoflurane before ischemia and during reperfusion protects against ischemia-reperfusion-induced injury in isolated rabbit lungs.

	Introduction

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Ischemia/reperfusion (IR)-induced lung injury is still a clinical problem, particularly after cardiopulmonary bypass or lung transplantation. The main problem in IR lung injury is dysfunction of the pulmonary vascular endothelium, manifested by pulmonary hypertension and increased vascular permeability. This results in pulmonary edema and impaired gas exchange. The effects of volatile anesthetics on IR-induced organ injury are controversial. Previous studies have reported that isoflurane protects the heart and liver against IR-induced injury (1,2). These studies indicate that isoflurane may also protect against IR-induced injury of the lung. In contrast, a recent study by Nielsen et al. (3) indicated that desflurane worsened thoracic aorta occlusion-reperfusion–induced lung injury. Theoretically, because there is no reason to believe that desflurane is unique in causing a detrimental effect, all halogenated anesthetics, including isoflurane, may cause similar detrimental effects on the injured lung (4).

In this study, we used an isolated rabbit lung model to investigate the effects of isoflurane on IR-induced lung injury by administering isoflurane before ischemia or during reperfusion. Because IR-induced lung injury is characterized by pulmonary hypertension and increased vascular permeability in vivo, we measured total pulmonary vascular resistance (Rt) and coefficient of filtration (Kfc) to determine serial changes in vascular permeability and to assess the integrity of the pulmonary vasculature in our isolated perfused lung preparation.

	Methods

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The experimental protocol was approved by Tottori University Laboratory Animal Care Committee. Female white Japanese rabbits weighing 2.0–2.5 kg were anesthetized with IV pentobarbital (25 mg/kg), followed by intramuscular ketamine (50 mg/kg). Heparin was injected IV (500 U/kg) before surgery. After local anesthesia was induced with 1% lidocaine, tracheostomy and sternotomy were performed. The rabbits' lungs were mechanically ventilated with a ventilator (Harvard 681; Harvard Apparatus, Natick, MA) using room air, set at a rate of 40 breaths/min, tidal volume of 6 mL/kg, and a positive end-expiratory pressure of 3 cm H₂O. The right common carotid artery was dissected, and a blood sample of 70 mL was collected. The pulmonary artery and left atrium were cannulated via the right and left ventricles. The heart, lungs, and mediastinal structures were removed en bloc and suspended via a counterbalanced force-displacement transducer (T1-200-240; Orientec, Tokyo, Japan) into a humidified, thermostatically controlled chamber (38°C) to monitor weight changes (Fig. 1). Mechanical ventilation of the lungs was also used at the same setting using a warm humidified gas mixture (21% O₂, 5% CO₂, balance N₂). Hematocrit of the perfusate was adjusted to approximately 15% by mixing autologous whole blood with physiological salt solution (PSS). PSS contained (in mM) NaCl 119, KCl 4.7, MgSO₄ 1.17, NaHCO₃ 22.61, KH₂PO₄ 1.18, and CaCl₂ 3.2. To each 100 mL of PSS stock solution, dextrose 100 mg, insulin 20 mU, and 3 g of bovine serum albumin (Sigma, Chemical Company, St. Louis, MO) were added. The pH of the perfusate was adjusted to 7.35–7.45 by adding sodium bicarbonate. Pulmonary arterial (Ppa) and venous (Ppv) pressures were continuously monitored by transducers connected to amplifiers (Model 2238; San-ei, Tokyo, Japan). The tip of a flow probe (Model FF-050T; Nihon Kohden, Tokyo, Japan) connected to an electromagnetic flowmeter (MFV-3100; Nihon Kohden) was placed in the perfusion circuit for continuous monitoring of blood flow (Q). A constant Ppa was established by maintaining a constant flow at 30 mL · min^-1 · kg^-1 by adjusting the level of arterial reservoir at the beginning of the experiment. Ppv was adjusted to 8 mmHg to ensure it exceeded the mean airway pressure. Consequently, the driving pressure remained constant throughout the experiment. Zero level was assigned to the bottom of the lung. All signals were digitized using an analog to digital converter (DigiData 1200; Axon Instruments, Foster City, CA) and were analyzed with commercially available software (Axograph ver. 3.0, Axon Instruments). Isoflurane (Abbott Laboratories, North Chicago, IL) was administered using an isoflurane vaporizer (Acoma, Tokyo, Japan). The concentration of isoflurane in the inspired gas was monitored with an anesthetic monitor (Capnomac Ultima^TM; Datex-Engstrom, Helsinki, Finland).

View larger version (28K): [in this window] [in a new window]   Figure 1. Experimental set up. PA = pulmonary artery, LA = left atrium, Paw = airway pressure, PAP = pulmonary artery pressure, LAP = left atrial pressure, FD = force displacement, PEEP = positive end-expiratory pressure. Arterial and venous reservoirs were elevated simultaneously by 6 cm H2O for measurement of the coefficient of filtration. Two pinch valves were simultaneously occluded for >5 s to obtain pulmonary capillary pressure. The pump continuously circulated the perfusate from the venous reservoir to the arterial reservoir to maintain a constant driving pressure.

Kfc was determined using the method described by Drake et al. (5). Briefly, after an isogravimetric state was achieved, i.e., no gain or loss of lung weight, the pulmonary capillary pressure (Ppc) was increased by elevating both arterial and venous reservoirs by 6 cm H₂O for 7 min, and the lung weight gain was recorded. The initial rapid lung weight gain (caused by vascular filling and distention) was followed by a slower rate of weight gain (caused by capillary filtration). We analyzed the rate of weight change (W/t) during a 3- to 6-min interval of increased Ppc. The initial rate of fluid filtration ([Wt/t]t = 0) was then calculated by extrapolating W/t to time 0. Kfc was calculated by dividing W/t at time 0 ([Wt/t]t = 0) by the change in Ppc (Ppc), then normalized using the baseline wet lung weight.

To determine Ppc, both arterial and venous lines were simultaneously occluded for >5 s with fast pinch valves (PK 0305-NO; Takasago, Tokyo, Japan). Ppa and Ppv converged to a certain level, which was defined as Ppc. Ppc was calculated as the difference between Ppc measured before and 7 min after elevated of the reservoir level. During Kfc determination, the positive pressure ventilation was interrupted, but a constant flow of 2 L/min mixed gas was administered at 3 cm H₂O airway pressure. Baseline wet lung weight was estimated at the end of the experiment by subtracting the weight of extrapulmonary tissues from the total weight of the lungs and extrapulmonary tissues, which was measured before perfusion.

The left lung was used to estimate the tissue W/D ratio. After recording the wet weight of the tissue sample, the lung was placed in a drying oven at 60°C for 2 wk and reweighed. W/D was calculated using the formula ([wet weight - dry weight]/dry weight).

The right lung was used for bronchoalveolar lavage (BAL) fluid preparation at the end of the experiment. Four aliquots (5 mL each) of isotonic sodium chloride solution were instilled separately through the trachea and drained. The collected fluid was centrifuged immediately at 250g and 4°C for 10 min. The supernatant was stored at -80°C until analysis. The concentration of nitrate + nitrate was determined by using a calorimetric assay kit (Cat#780001; Cayman Chemical Co., Ann Arbor, MI).

The lungs were allowed to equilibrate for 30 minutes to achieve an isogravimetric state. In the control (Cont) group (n = 6), the lungs were continuously perfused and ventilated for 120 min. In the ISO-Cont group (n = 6), 1 minimum alveolar anesthetic concentration (MAC) (1 MAC = 2.05% for rabbits) isoflurane was administered for 30 min during equilibration, then the lungs were continuously perfused and ventilated for 120 min. In the IR group (n = 6), ventilation and perfusion were interrupted (ischemia) after equilibration, and the lungs were maintained in the humidified chamber for 60 min. The lungs were reperfused and reventilated for 60 min after ischemia. In the ISO-IR group (n = 6), 30 min before ischemia, 1 MAC isoflurane was administered for 30 min, then ischemia was performed for 60 min, followed by 60 min reperfusion. In IR-ISO groups, reperfusion after 60 min ischemia was also accompanied by the administration of 1 MAC isoflurane (IR-ISO1 group, n = 6), or 2 MAC (IR-ISO2 group, n = 6) for 60 min.

Rt was determined at baseline (after equilibration); 5, 30, and 60 min after reperfusion in IR groups; and at the same intervals in the Cont and ISO-Cont groups. Kfc was determined at baseline and at the end of reperfusion or perfusion.

All data are presented as mean ± SEM. Within-group differences were analyzed by using one-way analysis of variance (ANOVA) with repeated measures (Statview 4.5; Abacus Concepts, Berkeley, CA). When statistical significance was observed by using ANOVA, a contrast test was performed for multiple comparisons (Super ANOVA; Abacus Concepts). Multiple samples of the same groups were analyzed by using ANOVA, followed by post hoc analysis with Bonferroni/Dunn method. Significance was determined at P < 0.05.

	Results

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Rt and Kfc were similar in all study groups at baseline.

Kfc significantly increased after reperfusion in the IR group (Fig. 2). The administration of isoflurane before and during reperfusion abrogated such an increase in Kfc.

View larger version (21K): [in this window] [in a new window]   Figure 2. Changes in the coefficient of filtration (Kfc). Data are mean ± SEM (n = 6 per group). *P < 0.05 versus baseline. #P < 0.01 versus other groups at the end of reperfusion.

View larger version (26K): [in this window] [in a new window]   Figure 3. Changes in total pulmonary vascular resistance (Rt). Data are mean ± SEM (n = 6 per group). = Cont group, • = IR group, = ISO-IR group, = ISO-Cont group, = IR-ISO1 group, = IR-ISO2 group. *P < 0.05 versus baseline. #P < 0.05 versus 5 min. !P < 0.05 versus IR group. !!P < 0.01 versus IR group.

View larger version (30K): [in this window] [in a new window]   Figure 4. Changes in lung wet to dry weight (W/D) ratio. Data are mean ± SEM (n = 6 per group). *P < 0.05 versus IR group.

	Discussion

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Our main findings are: (a) the administration of isoflurane (1 and 2 MAC) during reperfusion inhibited the increase in Kfc and W/D; (b) 2 MAC isoflurane inhibited the increase in Rt; and (c) the administration of isoflurane (1 MAC) before ischemia significantly attenuated the increase in Kfc, W/D, and Rt. These results indicate that isoflurane protected the lungs against IR-induced injury in our isolated lung preparation.

Studies have examined the effect of isoflurane on the heart and liver and have indicated that the volatile anesthetic exerts a protective effect on IR injury (1,2). In the isolated, blood-free, perfused liver, both 1 and 2 MAC isoflurane attenuates hypoxia-reoxygenation injury in rats (2). In a mouse model of multiple organ dysfunction syndrome, isoflurane (1.5%) attenuates lung inflammation and injury, probably due to modulation of the inflammatory response by volatile anesthetics (6). High concentrations of volatile anesthetics may induce toxic effects; halothane and isoflurane enhance the sensitivity of pulmonary artery endothelial cells to oxidant-mediated injury when administered at concentrations higher than 2.8% (3 MAC for halothane, 2.24 MAC for isoflurane in rats) (7). However, at lower halothane concentrations (0.4%, 0.5 MAC in rats), the volatile anesthetic exhibits a protective effect on H₂O₂-induced rat pulmonary artery endothelial cell injury (7). In contrast, Nielsen et al. (3) provided dirct evidence that desflurane increased IR-mediated pulmonary alveolar-capillary membrane permeability, but no difference in lung W/D was found among the different groups, which was attributed to a type 2 error (3). The reason for contradictory results between Nielsen et al.'s study and ours are not clear. However, differences in the study design and the use of different drugs may have contributed to the observed differences. For example, Nielsen et al. (3) used an in vivo rabbit model, in which lung injury was induced by thoracic aorta occlusion-reperfusion, but not pulmonary artery occlusion-reperfusion, and used protein level in BAL fluid as a marker for pulmonary alveolar-capillary membrane permeability.

Several techniques have been used to quantify microvascular injury caused by IR. The most reliable method is histological examination of the lung, which provides details on the site and extent of injury. However, this method often fails to adequately provide quantitative information about the functional status of the microvascular barrier. Although protein flux into airways and lung W/D are qualitative measures, they can be used in situ and likely yield directional changes when the magnitude of the change is large. Unfortunately, these variables are dependent on microvascular pressure; they increase when capillary pressure increases. In comparison, Kfc measured under isogravimetric condition is a more reliable and reproducible measure of lung injury in isolated dog, rabbit, ferret, rat, and guinea pig lungs (5,8). The measurement of Kfc has been reported in various IR studies and other isolated lung models to assess microvascular lung (8–10). A high Kfc reflects a high microvascular permeability. In our study, the results of Kfc were consistent with those of W/D. Thus, changes in both variables indicate that isoflurane abrogates an IR-induced increase in vascular permeability.

Although the mechanism of the protective effects of isoflurane was not determined in the present study, the administration of isoflurane may result in dilation of IR-constricted vessels. A low Rt reflects better blood flow, which is vital for the ischemic organs and results in improvement of oxygenation. Considering the protective role of nitric oxide (NO) in IR injury (11,12), we also investigated whether the protective effects of isoflurane are mediated by NO production in the lung. However, our results showed no significant difference in NO metabolites in BAL fluid among the different groups. Although we do not know how well the level of nitrate + nitrite in BAL fluid reflects the concentration of NO in the vicinity of pulmonary vascular endothelial cells, it is likely that the concentration of nitrite + nitrate in BAL fluid reasonably reflects the total amount of NO in the lung. NO is produced not only by the pulmonary vascular endothelium, but also by alveolar epithelial cells (13–15). In this regard, several studies have demonstrated that changes in endogenous NO parallel changes in nitrate and nitrite concentrations in BAL fluid (16,17). The present results suggest that the protective effect of isoflurane was not significantly mediated through NO production. Other possible explanations for isoflurane's protective effect against IR-induced injury are that it directly suppresses metabolism, depresses the utilization of adenosine triphosphate, or activates K_ATP channels. Furthermore, isoflurane reduces oxygen consumption in hypoxia-reoxygenated hepatic tissues (18) and directly preconditions the myocardium against infarction via activation of K_ATP channels (19). In the isolated perfused rat lungs, activation of K_ATP channels has been shown to protect against and reverse the endothelial damage associated with IR (20).

The main shortcoming of our study is the absence of histological evidence of lung injury. Although the isolated and perfused lung preparation is a reliable model by which to investigate the lungs under various pathophysiological conditions, the nature of the experimental set up also makes it difficult to extrapolate the findings to the clinical situation. Rt increased at 120 min from baseline in the Cont and ISO-Cont groups and this may represent one of the limitations of the present model. Furthermore, metabolites of isoflurane and their toxicity were not considered in the isolated lung model because isoflurane is primarily metabolized in the liver. Further studies using in vivo experiments, as well as clinical studies, are warranted.

In conclusion, we demonstrated that the administration of isoflurane before ischemia and during the reperfusion period attenuated IR-induced injury in isolated perfused rabbit lungs.

	Acknowledgments

 
We thank Dr. Naoto Okazaki for his assistance in the chemical analysis and Kfc measurement. The editorial assistance of Dr. K. G. Issa is also gratefully acknowledged.

	References

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