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CRITICAL CARE AND TRAUMA

Sevoflurane Pretreatment Inhibits Endotoxin-Induced Shock in Rats

Yoko Kidani, MD^*, Takumi Taniguchi, MD, Hiroko Kanakura, MD^*, Yasuhiro Takemoto, MD^*, Kazunobu Tsuda, MD^*, and Ken Yamamoto, MD^*

Departments of *Anesthesiology and Intensive Care Medicine and Emergency and Critical Care Medicine, Graduate School of Medical Science, Kanazawa University, Kanazawa, Japan

Address correspondence and reprint requests to Yoko Kidani, MD, Department of Anesthesiology and Intensive Care Medicine, Graduate School of Medical Science, Kanazawa University, 13-1 Takara-machi, Kanazawa 920-8641, Japan. Address e-mail to yousama{at}anesth.m.kanazawa-u.ac.jp.

	Abstract

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We examined the effects of sevoflurane pretreatment on mortality and inflammatory responses during endotoxin-induced shock. Rats were allocated randomly to 1 of 4 groups (n = 12 per group): an endotoxemia group, receiving IV Escherichia coli endotoxin (15 mg/kg over 2 min); a saline control group, receiving 0.9% saline (1.0 mL/kg); a sevoflurane-only group, receiving 2.4% sevoflurane for 30 min immediately before injection of 0.9% saline; and a sevoflurane pretreatment group, receiving 2.4% sevoflurane for 30 min immediately before injection of endotoxin. Hemodynamic variables, arterial blood gases, and plasma concentrations of tumor necrosis factor- and interleukin-6 were measured. The 8-h mortality rate was determined. Systolic arterial blood pressure and acid-base balance improved with sevoflurane pretreatment before induction of endotoxemia. Mortality rates 8 h after endotoxin injection were 83%, 8%, 0%, and 25% for the endotoxemia, saline control, sevoflurane-only, and sevoflurane pretreatment groups, respectively. Plasma cytokine concentrations were significantly larger in the endotoxemia group than in the other groups. Sevoflurane pretreatment inhibited inflammatory responses and decreased mortality in rats exposed to endotoxin.

	Introduction

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Endotoxemia, a major cause of systemic inflammatory response syndrome, is often resistant to intensive care (1). Although the pathophysiology of this state is not well defined, cytokines are considered important in mediating the associated cardiovascular disturbance. Circulating endotoxin induces activation of complement and release of cytokines such as tumor necrosis factor (TNF)- and interleukin (IL)-6, which, in turn, can promote infiltration of the lungs by leukocytes (2–4). Inflammatory mediators from these leukocytes can induce hypotension, metabolic acidosis, and tissue damage, eventually leading to organ dysfunction (5–7).

According to several reports, volatile anesthetics show a preconditioning effect in the ischemic heart (8–10). Recent investigations in vitro and in vivo have concluded that isoflurane pretreatment had antiinflammatory effects and also attenuated hypotension and myocardial dysfunction in endotoxemia and sepsis (11–14). Plachinta et al. (11) showed that pretreatment with isoflurane attenuated hypotension, acidosis, and increases in TNF- associated with endotoxin-induced inflammation in rats. Hayes et al. (12) reported that isoflurane pretreatment supported hemodynamic variables and leukocyte rolling velocities in rats exposed to endotoxemia. However, few studies have addressed these issues in sevoflurane treatment preceding endotoxemia and endotoxic shock. We therefore examined the effects of sevoflurane pretreatment on mortality and inflammatory responses during endotoxin-induced shock in rats.

	Methods

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Forty-eight male Wistar rats (12 ± 1 wk old, weighing 350 ± 20 g) were used in this study. The Animal Care Committee of our institution approved the experimental protocol, and care and handling of these animals were in accord with United States National Institutes of Health guidelines.

General Procedure
Rats were prepared as reported previously (8–13). Briefly, after intraperitoneal injection of pentobarbital sodium (30 mg/kg), ventilation was administered through a tracheotomy. The femoral artery was cannulated to monitor the arterial blood pressure and to draw blood samples. Ringer’s lactate solution containing a muscle relaxant (pancuronium bromide, 0.02 mg/mL) and pentobarbital sodium (0.5 mg/mL) was infused continuously at a rate of 10 mL kg^–1 h^–1 through the femoral vein cannula to attain a normovolemic state. Rats were connected to a pressure-controlled ventilator (Servo 900C; Siemens-Elma, Solna, Sweden), which delivered 100% oxygen at a frequency of 30 breaths/min with an inspiratory/expiratory ratio of 1:1. After this procedure, rats were rested for >30 min to allow the blood gases and hemodynamic variables to stabilize, followed by baseline readings of heart rate (HR) and systolic arterial blood pressure (SAP).

Experimental Protocol
After the baseline measurement, rats were allocated randomly to one of four groups, two of which received endotoxin.

Endotoxemia Group (n = 12)
Endotoxemia was induced by a bolus injection of lipopolysaccharide (10 mg/mL) derived from Escherichia coli 0111:B4 (Difco Laboratories, Detroit, MI), which was injected IV at a dose of 15 mg/kg over 2 min.

Saline Control Group (n = 12)
This group was not exposed to endotoxin, instead receiving an IV bolus injection of 0.9% saline (1.5 mL/kg).

Sevoflurane-Only Group (n = 12)
This group was not exposed to endotoxin, and received 2.4% sevoflurane for 30 min immediately before a bolus injection of 0.9% saline (1.5 mL/kg).

Sevoflurane Pretreatment Group (n = 12)
Endotoxemia was induced as for the endotoxemic group with lipopolysaccharide. Sevoflurane 2.4% was given for 30 min immediately before injection of lipopolysaccharide (15 mg/kg).

Rectal temperature was maintained between 36° and 38°C with the aid of a heating pad. Arterial blood samples (0.25 mL) were drawn 1, 3, and 5 h after endotoxin or saline injection for the measurement of arterial pH (pHa), arterial CO₂ tension (Paco₂), and arterial oxygen tension (Pao₂). Additional arterial blood samples (1.5 mL) were drawn for measurement of plasma cytokine concentrations at 2, 4, and 5 h after endotoxin or saline injection. A total amount of 5.5 mL of blood was drawn from each rat over 8 h. Cytokine concentrations (TNF- and IL-6) were measured using enzyme-linked immunosorbent assay kits (BioSource, Camarillo, CA). Lower limits of detection for TNF- and IL-6 were 4.2 and 6.5 pg/mL, respectively. The mortality rate was determined during an 8-h period after endotoxin or saline injection.

Statistical Analysis
Data are presented as the mean ± sd. Analysis of variance for repeated measures was used to compare hemodynamic and cytokine changes during the study. Differences among groups were analyzed with one-way analysis of variance followed by the Dunnett post hoc test. Comparisons of mortality rates among groups were made with the Kaplan-Meier and the Mantel-Cox methods. Statistical significance was defined as a P value < 0.05. Statistical analyses were performed using the StatView application (version 5.0; Abacus Concepts, Berkeley, CA).

	Results

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Hemodynamics and Mortality Rate
No significant differences were noted among groups in baseline HR or SAP (Fig. 1). Endotoxin injection reduced SAP in the endotoxemia group, but SAP did not decrease in the other groups. Mortality rates 8 h after endotoxin injection were 83%, 8%, 0%, and 25% for the endotoxemia, saline control, sevoflurane-only, and sevoflurane pretreatment groups, respectively (Fig. 2). The mortality rate for the endotoxemia group was significantly higher than for the other groups (P < 0.001).

View larger version (23K): [in this window] [in a new window]   Figure 1. The heart rate (top) and systolic arterial pressure (bottom) at baseline and after injection of endotoxin (mean ± sd). Closed circles = endotoxemia group, open circles = saline control group, closed squares = sevoflurane-only group, open squares = sevoflurane pretreatment group.

View larger version (17K): [in this window] [in a new window]   Figure 2. Survival curves for sevoflurane-only, saline control, sevoflurane pretreatment, and endotoxemia groups. *P < 0.05 versus endotoxemia group.

Plasma Cytokine Concentrations
All baseline values were similar for the four groups. The endotoxin injection increased the TNF- concentration in the endotoxemia and sevoflurane pretreatment groups, but the concentration was smaller in the sevoflurane pretreatment group than in the endotoxemia group (P < 0.05) (Fig. 3, top). Plasma IL-6 concentrations also increased in all groups (Fig. 3, bottom), but were significantly smaller in the sevoflurane pretreatment group than in the endotoxemia group (P < 0.05) (Fig. 3).

View larger version (20K): [in this window] [in a new window]   Figure 3. Changes of plasma tumor necrosis factor (TNF)- (top) and interleukin (IL)-6 (bottom) at baseline and after injection of endotoxin (mean ± sd). Closed circles = endotoxemia group, open circles = saline control group, closed squares = sevoflurane-only group, open squares = sevoflurane pretreatment group. *P < 0.05 versus endotoxemia group.

Blood Gases
Paco ₂ and Pao ₂ did not differ significantly among the 4 groups at any point during the experimental period (Table 1). The pHa was reduced in the endotoxemia group, but not in the other groups. Acid-base balance therefore was maintained better in the sevoflurane pretreatment group than in the endotoxemia group.

	Discussion

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Endotoxemia in the absence of sevoflurane pretreatment produced a decrease in SAP, an increase in plasma cytokine concentrations, and metabolic acidosis. Moreover, endotoxemia resulted in frequent mortality. In contrast, sevoflurane treatment before endotoxin exposure inhibited hypotension, production of cytokines, and metabolic acidosis, and also reduced the mortality rate. Thus, sevoflurane pretreatment improved the mortality rate and inhibited inflammatory responses in a rat endotoxin-shock model.

According to previous reports, volatile anesthetics, including sevoflurane and isoflurane, exert cardioprotective effects during ischemia and reperfusion in vitro and in vivo (8–10,15–19). Novalija et al. (18) showed that preconditioning with sevoflurane improved postischemic contractility in isolated hearts, and Toller et al. (19) found that sevoflurane reduced myocardial infarct size in dogs. However, few studies have addressed the effects of sevoflurane pretreatment during endotoxemia and endotoxic shock. The present study demonstrated that sevoflurane pretreatment improved the mortality rate and inhibited inflammatory responses in a rat model of endotoxin shock.

Inhibition by sevoflurane pretreatment of TNF- and IL-6 production after endotoxin injection is a finding of considerable interest. Circulating endotoxin induces release of cytokines such as TNF- and IL-6, which can produce hypotension and metabolic acidosis. Our study found that the increases in plasma concentrations of TNF- and IL-6 in the sevoflurane pretreatment group were significantly smaller than those in the endotoxemia group. Several investigators have reported the effects of sevoflurane pretreatment on cytokines. In vitro, Mitsuhata et al. (20) found that sevoflurane inhibited IL-1ß and TNF- production by human peripheral blood mononuclear cells, and Liu et al. (21) reported that sevoflurane administered at 1 minimum alveolar concentration before ischemia inhibited an increase in TNF- during ischemia-reperfusion-induced injury in isolated rat lungs. Our findings in vivo demonstrated that, here too, sevoflurane pretreatment inhibited cytokine responses to endotoxemia. These findings suggest that one of the mechanisms of the antiinflammatory effects of sevoflurane pretreatment may be down-regulation of cytokine production by macrophages and monocytes.

The present study could not elucidate details of the mechanisms underlying antiinflammatory effects of sevoflurane pretreatment. Zhong et al. (22) showed that sevoflurane preconditioning attenuated nuclear factor B (NF-B) activation and down-regulated NF-B-dependent inflammatory gene expression. Hu et al. (9) reported that sevoflurane pretreatment inhibited neutrophil activation and neutrophil-endothelial interaction. Further investigations are needed to clarify this point.

Two important questions that remain are whether sevoflurane pretreatment would have similar beneficial effects when given at different times after endotoxin administration and whether a dose-response relationship exists between sevoflurane and outcome. Here, too, answers await disclosure in further studies.

In summary, extremely frequent mortality rates, hypotension, acidosis, and increases in plasma cytokine concentrations after endotoxin injection in rats were attenuated by sevoflurane pretreatment. These findings indicate that sevoflurane pretreatment may inhibit inflammatory responses.

	Footnotes

 
Accepted for publication March 28, 2005.

	References

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