Isoflurane–Sevoflurane Administration before Ischemia Attenuates Ischemia–Reperfusion-induced Injury in Isolated Rat Lungs

The experimental protocol was approved by Tottori University Laboratory Animal Care Committee. Adult male Sprague-Dawley rats (weighing 300–350 g) were anesthetized with pentobarbital (20 mg/kg intraperitoneal) and ketamine (50 mg/kg intramuscular). After local anesthesia with 1.0% lidocaine, tracheostomy and sternotomy were performed. The lungs were mechanically ventilated with a ventilator (model 681; Harvard Apparatus, Natick, MA) using room air at a rate of 50 strokes/min, a tidal volume of 2.5 ml, and a positive end-expiratory pressure (PEEP) of 2 cm H²O until the cannulation of the pulmonary artery and left atrium was complete, after which the rat was ventilated in the same conditions with a warm humidified gas mixture of 21% O², 5% CO², and balance nitrogen. After sternotomy, heparin (100 U) was administered via  the right ventricle and allowed to circulate for approximately 3 min. Catheters were inserted through the right and left ventricles and secured in place with sutures. The lungs were perfused with bicarbonate-buffered physiologic salt solution. Physiologic salt solution contained 119 mM NaCl, 4.7 mM KCl, 1.17 mM MgSO⁴, 22.61 mM NaHCO³, 1.18 mM KH²PO⁴, and 3.2 mM CaCl². To each 100 ml of this stock solution, we added 100 mg dextrose, 20 mU insulin, and 5 g bovine serum albumin (Sigma, Chemical Co., St. Louis, MO). The pH of perfusate was adjusted to 7.35–7.45 by the addition of sodium bicarbonate. The lungs and heart were removed en bloc  and suspended from a counterbalanced force-displacement transducer (T1-200-240; Orientec, Tokyo, Japan) into a humidified chamber set at 37°C to monitor weight changes (fig. 1). The first 75 ml perfusate, which contained large amounts of residual blood cells and plasma, was discarded, and an additional 50 ml volume of perfusate was then used for recirculation.

Pulmonary arterial (Ppa) and venous (Ppv) pressures were continuously monitored by transducers connected to amplifiers (model 2238; San-ei, Tokyo, Japan). 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). Pulmonary venous pressure was adjusted to 5 mmHg to ensure that pulmonary venous pressure exceeded the mean airway pressure, then pulmonary arterial pressure was adjusted by maintaining the flow at 0.04 ml/g body weight/min by adjustment of the arterial reservoir level at the beginning of the experiment. Consequently, the driving pressure was kept constant throughout the experiment. Zero level for pressures was referenced as the bottom of the lung in the chamber. All signals were digitized using an analog-to-digital converter (DigiData 1200; Axon Instruments, Foster City, CA) and analyzed using a commercially available software (Axogragh version 3.0; Axon Instruments). Isoflurane (Abbott Laboratories, North Chicago, IL) or sevoflurane (Maruishi Pharmaceutical, Osaka, Japan) was administered using isoflurane (Acoma, Tokyo, Japan) or sevoflurane (Penlon; Penlon Limited, Abing, Oxon, UK) vaporizers. The concentration of isoflurane and sevoflurane in inspired gas was monitored using anesthetic agent monitor (Capnomac; Datex Instrumenttarium Corp., Helsinki, Finland).

Total pulmonary vascular resistance (Rt) was calculated using the following formula: Rt = (Ppa − Ppv)/Q.

The Kfc was determined using the method described by Drake et al.  11Briefly, 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 the arterial and the 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 distension) was followed by a slower rate of weight gain (caused by capillary filtration). The logarithm of the rate of lung weight gain (ΔW/Δt) was plotted as a function of time. We analyzed the ΔW/Δt during a 3- to 6-min interval of increased Ppc. The initial rate of fluid filtration [(ΔW/Δt)t = 0] was then calculated by extrapolating ΔW/Δt to time 0. Kfc was calculated by dividing ΔW/Δt at time 0 [(ΔW/Δt)t = 0 ] by changes in Ppc and normalized using the baseline wet lung weight and expressed in ml · min⁻¹· mmHg⁻¹· 100 g⁻¹lung tissue.

To determine Ppc, arterial and venous lines both were occluded simultaneously for more than 5 s with fast-pinch valves (PK 0305-NO; Takasago, Tokyo, Japan); pulmonary arterial pressure and pulmonary venous pressure converged to a certain level, which was defined as Ppc. Change in Ppc was calculated as the difference between Ppc measured before and 7 min after elevation of the reservoir level. During Kfc determination, the ventilator was switched off, but a constant flow of 2 l/min of mixed gas was administered at 2 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 lungs were used to estimate the tissue wet-to-dry weight ratio. After recording the wet weight of the lung tissue, the lungs were placed in a drying oven at 60°C for 2 weeks and reweighed. Wet-to-dry weight ratio was calculated using the formula: (wet weight − dry weight)/dry weight.

Samples of perfusate (2.5 ml each) were obtained immediately after 30 min of equilibration, at 60 min after reperfusion in IR groups, and at these same intervals in the control group. The collected perfusate was centrifuged immediately at 250 g  for 10 min at 4°C. The supernatant was divided into several aliquots and stored at −80°C until analysis. The concentrations of NO metabolites (sum of nitrate and nitrite) were determined using a calorimetric assay kit (Cat#7 80001; Cayman Chemical Company, Ann Arbor, MI). 12TNF-α level was measured by using the rat TNF-α enzyme-linked immunosorbent assay kit (ELISA; Biosource International, Inc. CA). LDH activity was measured using a modification of the colorimetric method 13at 37°C. Changes in LDH activity and TNF-α in the perfusate were expressed as rate of increase = (end value − baseline value)/baseline value.

The lungs were allowed to equilibrate for 30 min to achieve an isogravimetric state. Lung preparations were designated into four groups. The total duration of the experiment was similar in each group. In the control group (n = 6), 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 for 60 min in the humidified chamber while the airway was kept open. The lungs were reperfused and reventilated for 60 min after ischemia. In the isoflurane (ISO)-IR group (n = 6), 1 minimum alveolar concentration (MAC) isoflurane (1.38%) was first administered for 30 min, followed by ischemia for 60 min, and later the preparation was perfused for 60 min. A similar protocol was used in the sevoflurane (SEVO)-IR group (n = 6), except that isoflurane was replaced with 1 MAC sevoflurane (2.11%).

The Kfc was determined at baseline (immediately before ischemia), 30, and 60 min after reperfusion in IR groups and at these same intervals in the control group. Rt was determined at baseline and 60 min after reperfusion in IR groups and at these same intervals in the control group.

Data are presented as the mean ± SD. Within-group differences were analyzed using one-way analysis of variance using repeated measures (Statview 4.5; Abacus Concepts, Berkeley, CA). Multiple samples of the same time intervals were analyzed using one-way analysis of variance. Changes in LDH activity, TNF-α, NO metabolites in the perfusate, and the wet-to-dry weight ratio were evaluated using one-way analysis of variance. The Bonferroni–Dunn test was used for post hoc  comparisons. Significance was determined when P  was less than 0.05.

Total pulmonary vascular resistance significantly increased at the end of the experiment in all groups, compared with baseline, from 279 ± 155 mmHg · l⁻¹· min⁻¹to 381 ± 197 mmHg · l⁻¹· min⁻¹in the control group (P < 0.05), from 273 ± 98 mmHg · l⁻¹· min⁻¹to 395 ± 156 mmHg · l⁻¹· min⁻¹in the IR group (P < 0.05), from 255 ± 103 mmHg · l⁻¹· min⁻¹to 432 ± 212 mmHg · l⁻¹· min⁻¹in the ISO-IR group (P < 0.05), and from 287 ± 119 mmHg · l⁻¹· min⁻¹to 446 ± 147 mmHg · l⁻¹· min⁻¹in the SEVO-IR group (P < 0.05). There were no significant differences among groups at both the baseline and the end of the experiment.

The level of Kfc (an index of microvascular permeability) at baseline was similar in all groups. Ischemia followed by reperfusion caused a significant increase (twofold at 30 min of reperfusion;P < 0.01) in microvascular permeability (Kfc) in the IR group, relative to the control group (fig. 2). Kfc further increased (sevenfold) at 60 min of reperfusion in the IR group (P < 0.01). Preadministration of 1 MAC isoflurane or sevoflurane prevented the deterioration in vascular permeability after IR.

The lung wet-to-dry weight ratio was calculated to estimate lung fluid content. IR significantly increased the lung wet-to-dry weight ratio in the IR group. In contrast, preadministration of isoflurane or sevoflurane abrogated the effect of IR on the lung wet-to-dry weight ratio (ISO-IR and SEVO-IR groups;fig. 3).

We used the release of LDH into the perfusate as an estimate of cell damage during the experiment. Figure 4shows that the rate of increase of LDH activity was significantly higher in the perfusate of the IR group compared with the control group. In contrast, preadministration of isoflurane or sevoflurane significantly inhibited the rate of increase of LDH activity. Measurement of NO metabolites showed a significantly low level of these metabolites in the perfusate at the end of reperfusion in the IR group relative to the control group. In contrast, isoflurane or sevoflurane significantly inhibited the decrease in NO metabolites (ISO-IR and SEVO-IR groups;fig. 5) at the end of reperfusion. The results of evaluation of the potential mechanism of the protective effects of isoflurane or sevoflurane against IR lung injury are shown in figure 6. IR significantly increased the rate of increase of TNF-α release in the IR group compared with the control group. Preadministration of isoflurane or sevoflurane significantly inhibited the rate of increase of TNF-α in the perfusate.

The main finding of the current study was that isoflurane and sevoflurane protected against lung injury induced by IR in an isolated rat lung model. Therefore, administration of 1 MAC isoflurane or sevoflurane before ischemia significantly attenuated the increase in Kfc (fig. 2) and wet-to-dry weight ratio (fig. 3), inhibited the release of LDH (fig. 4) and TNF-α (fig. 6), and reduced the production of NO metabolites (fig. 5) in the perfusate after IR.

The protective effect of volatile anesthetics against IR-induced injury has been described previously in other important organs (heart, brain, among others) when the volatile anesthetics were administered before ischemia 4,7or during early reperfusion, 5or when both were administered before ischemia and during reperfusion. 6In isolated blood-free perfused livers, isoflurane, administered during ischemia and reperfusion, attenuated hypoxia–reoxygenation injury in rats. 8Isoflurane, similar to halothane, administered before, during, and immediately after hypothermic preservation markedly improved cardiac perfusion and function. 14Sevoflurane, a new volatile anesthetic used clinically in many centers, also protects the heart and brain against IR-induced injury. 6,15 

Although the effects of volatile anesthetics on IR-induced lung injury remain unclear, in a mouse model of multiple organ dysfunction syndrome, isoflurane (1.5%) attenuated lung inflammation and injury, probably by modulating the inflammatory response. 16Halothane and isoflurane could protect against oxidant injury in aortic endothelial cells by amelioration of the toxic increase in cytosolic calcium. 17In an isolated rabbit lung model, isoflurane could attenuate IR-induced injury. 9In contrast to these findings, Nielsen et al.  18,19recently provided evidence that indicated that volatile anesthetics (halothane 18and desflurane 19) have detrimental effects on IR injury in the liver 18and lung 19induced by thoracic aorta occlusion–reperfusion in a rabbit model. Although no differences in lung wet-to-dry ratio were found among groups, the findings of Nielsen et al.  19raised an important issue regarding the effects of volatile anesthetics on IR-mediated lung injury. These studies are, however, not directly comparable with ours because of differences in the experimental design in which an in vivo  animal model was used and the lung injury was induced by reperfusion of the liver and intestines by thoracic aorta occlusion–reperfusion. The differences in the study design and the use of different models may have contributed to the observed differences in the results.

In contrast with our previous study in the rabbit, 9a different anesthetic (sevoflurane), species (rat), and lung injury indexes (LDH, NO metabolites) were used, and the possible mechanism (role of TNF-α) were investigated in the current study. IR lung injury, directly induced by IR of the isolated lungs, was evident by the increased Kfc and wet-to-dry weight ratio and the changes in LDH activity and NO metabolites in the perfusate. Kfc measured during isogravimetric conditions is a reliable and reproducible measure of lung injury in isolated animal lungs. 11,20–22A high Kfc means augmented microvascular permeability. In the current study, the results of Kfc were consistent with those of the wet-to-dry weight ratio, which indicates that isoflurane and sevoflurane could inhibit IR-induced increase in vascular permeability. Our findings of changes in LDH activity and NO metabolites in the perfusate further confirm that IR lung injury was induced in our in vitro  model. Reduced levels of NO metabolites in the perfusate noted in the current study reflected a low production of NO, which was probably a result of dysfunction of pulmonary endothelial cells caused by IR. Administration of isoflurane or sevoflurane before ischemia successfully protected the lung from IR injury, as indicated by the Kfc, the wet-to-dry weight ratio, LDH activity, and the NO metabolites in the perfusate.

As discussed previously, we demonstrated that volatile anesthetics reduce lung injury. However, whether this is because of prevention of changes or whether the volatile anesthetic itself produces some off-setting decrease in these markers is not clear. It is possible that the effect of volatile anesthetics is the opposite of that of IR, resulting in a net benefit, rather than being caused by a specific inhibition of certain pathologic processes. To confirm this conclusion, a supplementary experiment in which control lungs are ventilated with volatile anesthetic but not subjected to ischemia is necessary. We performed such experiment by administration of 1 MAC isoflurane followed by 120 min perfusion (ISO-control group, n = 5). No significant difference in currently used markers was found between the ISO-control group and the control group. In the ISO-control, Kfc was 0.39 ± 0.23 ml · min⁻¹· mmHg⁻¹· 100 g⁻¹at baseline and 0.45 ± 0.15 ml · min⁻¹· mmHg⁻¹· 100 g⁻¹at the end of perfusion (P = 0.733). Wet-to-dry ratio was 7.00 ± 0.97. The rates of increase of LDH activity and TNF-α were 0.58 ± 0.21 and 9.50 ± 5.25, respectively. The sum of NO metabolites in the perfusate was 3.93 ± 0.16 μM at baseline and 4.07 ± 0.34 μM at the end of perfusion (P = 0.067). These results showed that the volatile anesthetic itself does not produce any off-setting decrease in the currently used markers.

Although the precise mechanisms of IR-induced organ injury are still being investigated, recent studies indicate that TNF-α is an essential component in some models of lung injury. 1,23TNF-α was significantly released after IR. Furthermore, the damage associated with IR was totally eliminated by pretreating animals with a specific anti–TNF-α antibody. 1Significant elevation of local tissue levels of TNF-α was also observed after reperfusion after prolonged pulmonary ischemia. 24TNF up-regulates intercellular adhesion molecule 1 (ICAM-1), which is important in neutrophil-dependent lung and liver injury associated with hepatic IR in the rat. 2In the current study, the rate of increase of TNF-α in the perfusate was significantly increased after IR, whereas preadministration of isoflurane or sevoflurane significantly inhibited such an increase. These results suggest that the protective effect of isoflurane and sevoflurane against IR-induced lung injury may be mediated through inhibition of TNF-α release. Previous studies reported that volatile anesthetics could reduce postischemic adhesion of neutrophils in the coronary system in an IR heart model, 25inhibit neutrophil migration 26and generation of oxygen radicals by inflammatory cells, 27and inhibit the release of TNF-α from cultured human peripheral mononuclear cells. 10However, because our rat model is devoid of circulating neutrophils and free of specific markers of neutrophil activation, it is difficult to discern the source of TNF-α; however, it is possible that the resident neutrophils may be activated by the endothelial elaboration of TNF-α after ischemia or cause further elaboration of TNF-α by further injury of the reperfused endothelium. Considered together, further studies are warranted to investigate the exact role of TNF-α.

Considering that the volatile anesthetics are also excellent pulmonary vasodilators, the observed protective effect may represent changes secondary to changes in vascular smooth muscle tone; but no significant Rt difference was found among the groups at baseline or the end of the experiment in the current buffer-perfused lung model. Other possible explanations for the protective effects of isoflurane and sevoflurane on IR-induced lung injury include suppression of metabolism or depression of the use of adenosine triphosphate (ATP), and activation of potassium adenosine triphosphate channels. Isoflurane can decrease oxygen consumption in the hypoxia-reoxygenated liver 28and directly preconditions the myocardium against infarction via  activation of potassium adenosine triphosphate channels. 29Further studies are warranted.

The main shortcoming of the current study is the lack of any histologic evidence, which confirms the presence of lung injury. Although the isolated and perfused lung preparation is a suitable model for investigating the pulmonary response to injury and other pathophysiologic conditions, such an isolated lung model does not show many features associated with actual lung injury, and the artificial features also make it difficult to extrapolate the findings to the clinical situation. As we noticed in the current study, Rt significantly increased in all groups at the end of the experiments. Furthermore, the effects of potentially toxic metabolites of volatile anesthetics could not be evaluated in the isolated lung model because isoflurane and sevoflurane are primarily metabolized in the liver. Further studies using in vivo  experimental preparations, in addition to clinical studies, are warranted.

In conclusion, we demonstrated that isoflurane and sevoflurane, when administered before ischemia, exhibit beneficial effects manifested by attenuation of IR injury in the isolated perfused rat lungs.

The authors thank Dr. Naoto Okazaki for assistance in the chemical analysis and Kfc measurement and Dr. F. G. Issa for editing assistance.