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CRITICAL CARE AND TRAUMA

Propofol Attenuates the Decrease of Dynamic Compliance and Water Content in the Lung by Decreasing Oxidative Radicals Released from the Reperfused Liver

Kuang-Cheng Chan, MD^*, Chen-Jung Lin, MD^*, Po-Huang Lee, MD, PhD, Chau-Fong Chen, PhD, Yih-Loong Lai, PhD, Wei-Zen Sun, MD^*, and Ya-Jung Cheng, MD, PhD^*

From the Departments of *Anesthesiology, Surgery, National Taiwan University Hospital, National Taiwan University; and Department of Physiology, College of Medicine, National Taiwan University, Taipei, Taiwan.

Address correspondence and reprint requests to Dr. Ya-Jung Cheng, Department of Anesthesiology, National Taiwan University Hospital, 7, Chung-Shan S. Rd., Taipei, Taiwan. Address e-mail address to chyj888{at}anesth.mc.ntu.edu.tw.

	Abstract

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BACKGROUND: Remote pulmonary injuries after hepatic reperfusion are frequently caused by reactive oxygen species (ROS)-induced damage. The choice of anesthetics may affect the balance between oxidants and antioxidants, and propofol, a commonly used anesthetic, has an antioxidant effect. In this study, we developed a model to study pulmonary function with hepatic ischemia/reperfusion (I/R) manipulation, with the aim of defining remote pulmonary dysfunction after hepatic reperfusion and determining if propofol affects this dysfunction by altering ROS production from the liver or lungs.

METHODS: Adult male rats weighing 160–250 g were randomly divided into four groups according to the type of surgery (sham or I/R) and the anesthetic administered (pentobarbital or propofol). To induce I/R, the portal vein and hepatic artery to the left and medial lobes of the liver were clamped. All of the measurements were done after 5 h of reperfusion, after 45 min of ischemia. Pulmonary function after hepatic I/R was determined by dynamic compliance, resistance and wet-to-dry ratio, and by histopathology. Hepato-cellular injuries were confirmed by alanine aminotransferase, whereas ROS production was measured from the inferior vena cava, jugular vein, and carotid artery. Products of lipid peroxidation, thiobarbiturate acid reactive substances and malondialdehyde, were measured in lung and hepatic tissues.

RESULTS: Remote lung injury after hepatic I/R was shown by a significant decrease of Cdyn, and increases in resistance and the wet-to-dry ratio. ROS production was significantly increased and was highest in samples from the inferior vena cava. Thiobarbiturate acid reactive substances and malondialdehyde in the liver and serum alanine aminotransferase were significantly increased only in the I/R+pentobarbital group. All of the changes were significantly attenuated in the I/R+ propofol group (P = 0.05). With propofol infusion, there was decreased ROS production from the reperfused liver, with less hepato-cellular injury, followed by well-maintained pulmonary function.

CONCLUSION: Remote pulmonary dysfunction and reperfusion injury in the liver were demonstrated in our rat model, as well as massive ROS production and lipid peroxidation. Propofol infusion attenuated remote pulmonary injury by lessening oxidative injury from the reperfused liver.

	Introduction

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The occurrence of postoperative pulmonary complications after hepatic reperfusion, such as in patients undergoing liver transplantation, is a major concern in the intensive care unit. Not only neutrophil infiltration, but also oxidative injuries, have been demonstrated after intraoperative hepatic ischemia/reperfusion (I/R) management.1–4 Previous studies have shown that reactive oxygen species (ROS) play a major role in the ensuing damage, although I/R-induced remote organ injury is a complex and multifactorial process.4,5

Methods to reduce ROS generation, such as ischemic preconditioning, attenuate both liver and lung damage after hepatic I/R.6,7 Considering that intraoperative ROS production occurs after hepatic reperfusion, the choice of anesthetics may alter the magnitude of ROS production and the antioxidant capacity. Of the frequently applied anesthetics, propofol (2,6-diisopropylphenol) has been reported to exert greater antioxidant effects8–10 than other anesthetics.11,12 In terms of direct reperfusion injury to the lungs, propofol has also demonstrated to attenuate oxidant-induced acute lung injury in an isolated-perfused rabbit lung model.13 However, there are few investigations concerning the effects of different anesthetics on remote pulmonary injuries after hepatic reperfusion.

In this study, the respiratory functions (dynamic lung compliance [Cdyn]) and edema formation) and oxidative injury (ROS production and lipid peroxidation) were investigated by an I/R maneuver of the liver in a rat model. The goals of this study were: 1) to examine remote respiratory function after hepatic I/R and 2) to investigate whether propofol infusion attenuates remote pulmonary injury by affecting the oxidative/antioxidative balance of the liver or lungs.

	MATERIALS AND METHODS

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Animals
Adult male specific pathogen-free Sprague-Dawley rats weighing 160–250 g were obtained from the National Laboratory Breeding and Research Center (Taipei, Taiwan). The experimental protocol was approved by the Laboratory Animal Care Committee of the National Taiwan University College of Medicine.

Preparation of the Hepatic I/R Injury Model
All of the rats were weighed and anesthetized by intraperitoneal injection of sodium pentobarbital (35 mg/kg of body weight). After a suitable level of anesthesia had been attained, the trachea was intubated to maintain a patent airway, while the right jugular vein was catheterized for infusion of saline and drugs. The right carotid artery was catheterized for continuous measurement of systemic blood pressure and for blood withdrawal.

A rat model of lobar, rather than total, hepatic I/R was used as previously described.14–16 Briefly, a noncrushing microvascular clip was placed across the branches of the portal vein and hepatic artery to the left lateral and median lobes for 45 min. The clip was removed to allow reperfusion for 5 h. This model induced a severe ischemic insult to the liver without inducing mesenteric venous hypertension or subsequent bacterial translocation into the portal venous blood. Sham-operated control animals were treated in the same fashion, with the omission of vascular occlusion.

Experimental Design
The experimental animals were randomly divided into four groups (n = 15/group): Group 1, sham operation and maintained with pentobarbital sedation (Sham+Pen); Group 2, hepatic I/R and maintained with pentobarbital sedation (I/R+Pen); Group 3, sham operation and maintained with propofol sedation (Sham+Pro); and Group 4, hepatic I/R and maintained with propofol sedation (I/R+Pro). Anesthesia was maintained by propofol (25 mg · kg^–1 · h^–1) and pentobarbital (5 mg · kg^–1 · h^–1) infusion soon after jugular venous cannulation. Hepatic ischemia was induced after 40 min of infusion to allow for stabilization of our model. At the end of the experiment, the animals were killed with an overdose of either pentobarbital or propofol, according to group, and tissue samples were collected. We did not catheterize the inferior vena cava (IVC) for sampling. Rather, the blood for ROS measurement was drawn directly from the IVC with a syringe at 5 h after reperfusion, before the rat was killed.

Respiratory Functions
The animals breathed spontaneously in room air and were placed inside a whole-body plethysmograph (Buxco), with catheters connected to a transducer for measuring arterial blood pressure. A differential pressure transducer (Transpac 42584, Hospira) for measuring pressure difference across a wire mesh screen was placed on the wall of the plethysmograph and was used to measure airflow. The airflow signal was integrated to a signal proportional to volume.17 Esophageal pressure was measured with a differential pressure transducer (Transpac 42584, Hospira) and was used as the transpulmonary pressure.18 Volume, airflow, and transpulmonary pressure signals were monitored and displayed on a data acquisition system installed on a personal computer. Cdyn, mL/cm H₂O, and lung resistance (RL, cm H₂O, s/mL) were measured according to the methods of Amdur and Mead19 at baseline (before induction of I/R) and at 1, 3, and 5 h after reperfusion.

Edema Formation
Pulmonary edema was estimated by an increase in the wet-to-dry (W/D) weight ratio of the lungs.20

Measurement of Serum Liver Enzymes
Blood samples for measurement of serum alanine aminotransferase (ALT) were obtained via the indwelling venous catheter. Serum ALT was quantified using standard clinical automated analysis.

Measurement of ROS
Blood samples were immediately wrapped in aluminum foil and kept on ice until chemiluminescence (CL) measurement, which was usually performed within 2 h.21,22 Immediately before CL measurement, 0.1 mL phosphate-buffered saline (pH 7.4) was added per 0.2 mL blood sample. CL was measured in a completely dark chamber of the CL analyzing system. After 100 s of background level determination, 1.0 mL of 0.1 mM lucigenin in phosphate-buffered saline (pH 7.4) was injected into the sample. CL was continuously monitored for an additional 300 s. The assay was performed in duplicate for each sample and was expressed as CL counts per 10 s for whole blood CL.

Tissue Malondialdehyde (MDA) and Thiobarbiturate Acid Reactive Substances (TBARS) Measurements
Lipid peroxidation is used as an indirect measure of oxidative injury induced by ROS.23 Lipid peroxidation in liver and lung samples is determined by the thiobarbiturate reaction, which measures the formation of TBARS and MDA.7,24 TBARS is frequently used to screen the oxidative products, but it can also be generated during an assay from false chromogens and real chromogens. Thus TBARS measured in a sample will differ according to the assay condition used. MDA is thought to be the more reliable product of lipid peroxidation. In human plasma in most studies, MDA values are far lower than values obtained by TBARS tests.

Histologic Examination
Lung and liver blocks were embedded in paraffin; 5 µm sections were cut, and stained with hematoxylin and eosin. Slides were examined in a blinded manner.

Data Analysis
All of the data are presented as mean ± sem. The means of different groups were compared using one-way analysis of variance. The Student-Neuman-Keuls post hoc tests were applied for establishing differences between two groups. Significance was determined at the 5% level (P < 0.05).

	RESULTS

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There were no significant differences in body weight and arterial blood pressure among the three groups throughout the experiment.

Changes in Lung Functions
As shown in Figure 1, Cdyn was significantly decreased in the I/R+Pen group, whereas RL increased significantly at 5 h after hepatic reperfusion. The Cdyn and RL of the I/R+Pro group were comparable to those of the Sham group (control). The W/D ratio was significantly increased only in the I/R+Pen group, which also showed pulmonary edema histologically (Fig. 2). The W/D ratio was comparable among the Sham+Pen, Sham+Pro, and I/R+Pro groups (Table 1).

View larger version (11K): [in this window] [in a new window]   Figure 1. (A) Changes of dynamic compliance and (B) changes of lung resistance in the experimental animals. Values are expressed as means ± sem (% baseline). *P < 0.05, compared to baseline. **P < 0.01, compared to baseline. #P < 0.01, compared among groups. (Sham: sham operated group; I/R: ischemia and reperfusion group; Pen: sodium pentobarbital; Pro: propofol).

View larger version (109K): [in this window] [in a new window]   Figure 2. An example of lung histopathology in the following experimental animal groups: (A) Sham+Pentobarbital: normal alveolar expansion and intact alveolar and bronchiolar structure. (B) Ischemia/ reperfusion (I/R)+ Pentobarbital: widespread edema, intense neutrophil infiltration, disruption of alveolar and bronchiolar epithelial cells and hemorrhage. (C) Sham+Propofol: nearly normal lung tissue. (D) I/R+Propofol: mild neutrophil infiltration and slight thickening of alveolar walls.

Hepato-Cellular Damage
As shown in Table 1, plasma ALT activities increased significantly in both I/R groups compared with those in the sham groups, which were within normal limit. In the I/R groups, ALT activity was significantly lower in the I/R+Pro group than in the I/R+Pen group.

ROS Production
As shown in Table 2, there was a significant increase in ROS production from the IVC sampling in the I/R+Pen group. ROS production in the jugular vein and carotid artery were not significant but higher in both I/R groups.

Lipid Peroxidation in the Liver and Lungs
As shown in Table 3 and Table 4, the MDA and TBARS levels of the liver, including ischemic and nonischemic lobes, were significantly increased in the I/R+Pen group but not in the other three groups. Nonsignificant increases of MDA and TBARS were shown in lung tissues of the I/R+Pen group.

	DISCUSSION

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The main results of this study are: 1) hepatic reperfusion induced a significant deterioration of lung functions, including Cdyn, RL, and edema formation; and 2) propofol infusion attenuated remote pulmonary effects by decreasing ROS production from hepatic reperfusion. Although previous studies of hepatic reperfusion-induced lung injuries were confirmed,16 this is the first in vivo study that demonstrated remote pulmonary injuries by measuring Cdyn and RL, a reliable functional change detected by traditional body plethysmograph.19 In this rat model, remote lung dysfunction was found 5 h after hepatic reperfusion and was attenuated by propofol infusion. In humans, remote pulmonary injuries often appear 12–24 h after liver transplantation.2

A significant increase of lung W/D ratio suggestive of hepatic I/R-induced pulmonary edema occurred only in the I/R+Pen group. Comparable lung W/D ratio data were obtained in both sham and I/R+Pro groups. Due to comparable hemodynamic stability, transfusion, and fluid infusion, the increase of lung water after hepatic I/R may have been due to a significant increase in vascular permeability1 in this study.

ROS production was measured at 5 h after hepatic reperfusion and showed significant lung injury. Although it may not have reached peak value in the I/R response, a significant increase in ROS production was found in both I/R groups, but was reduced significantly by propofol infusion. ROS production in the I/R+Pen group was highest in the IVC, then the jugular vein, and then the carotid artery. The different levels of ROS production revealed that massive amounts of ROS were released from the reperfused liver, and were diluted in the jugular vein and the circulated carotid artery. ROS is possibly further cleared during circulation through the lungs, not only through systemic antioxidant effects but also by antioxidants in the lungs.

Although ROS production increased significantly after hepatic I/R in the I/R+Pen group, propofol infusion prevented this significant increase. Apparently, propofol infusion attenuates remote pulmonary injury by decreasing ROS production from reperfused liver, although this production was still higher in the I/R+Pro group compared to both sham groups. The associated lipid peroxidation that follows is also significantly decreased by propofol infusion. Thus, propofol attenuates pulmonary dysfunction by decreasing free radicals in the pulmonary circulation, even with a significant increase in hepatic enzymes.

Oxidative injury results from an overwhelming production of ROS that cannot be neutralized by antioxidants. It is therefore important to evaluate oxidative injury in the liver and lungs. In the hepatic I/R+Pen group, moderate but nonsignificant increases in pulmonary MDA and TBARS were shown. MDA and TBARS were highest in the ischemic part of the reperfused liver, although they were also significantly high in the nonischemic part. Changes in lipid peroxidation revealed the severity of ROS-induced injuries in each organ. In partial clamping of the liver, it is not only the ischemic part of the liver but also the nonischemic part that bears oxidative injuries. Reperfusion has been shown to affect hepatic function adversely because it induces the release of ROS from endothelium and neutrophils, and activates Kupffer cells. Both of these induce inflammatory mediators, such as tumor necrosis factor- and interleukin 1, which cause increased vascular permeability and accumulation of activated neutrophils.

Similar to our study, a previous study showed that partial hepatic IR resulted in damage in both ischemic and nonischemic liver.25 Although nonischemic liver did not undergo an ischemic event, circulating ROS may have played a role in the injury of nonischemic liver. An antioxidant effect of propofol in our study neutralized circulating ROS, thus lessening the ensuing liver damage. Based on our results, propofol infusion would be beneficial by decreasing oxidative injuries to the lungs and the liver.

In previous studies, the extent of remote lung damage was associated with the magnitude of liver injuries and circulating xanthine oxidase (XO) activity.5 Concerning oxidative stress, ROS was the first mediator released from the reperfused organ, but it was seldom reported because of the need for immediate tests.22 XO was not measured in this study, but if the initial massive ROS production was decreased, XO activity might also have been less.

In conclusion, this study showed that remote pulmonary dysfunction occurred 5 h after de-clamping of the hepatic vasculature in an ischemic liver, accompanied by a significant increase in hepatic enzymes and ROS production from the liver. Propofol infusion was shown to lessen remote pulmonary dysfunction through an initial reduction of ROS production from the reperfused liver.

	ACKNOWLEDGMENTS

 
The authors thank Dr. Chia-Tung Shun for expert pathological assistance.

	Footnotes

 
Accepted for publication May 23, 2008.

Supported by National Taiwan University Hospital Research Grant NTUH94M12.

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