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Anesth Analg 2001;92:437-441
© 2001 International Anesthesia Research Society

CRITICAL CARE AND TRAUMA

Propofol Depressed Neutrophil Hydrogen Peroxide Production More Than Midazolam, Whereas Adhesion Molecule Expression was Minimally Affected by both Anesthetics in Rats with Abdominal Sepsis

Takefumi Inada, MD*, Shoichiro Taniuchi, MD{dagger}, Koh Shingu, MD*, Yohnosuke Kobayashi, MD{dagger}, Junichi Fujisawa, PhD{ddagger}, and Shin-ichi Nakao, MD*

Departments of *Anesthesiology, {dagger}Pediatrics, and {ddagger}Microbiology, Kansai Medical University Hospital, 10-15 Fumizonocho, Moriguchi, Osaka 570-8507, Japan

Address correspondence and reprint requests to Takefumi Inada, MD, Department of Anesthesiology, Kansai Medical University Hospital, 10-15 Fumizonocho, Moriguchi, Osaka 570-8507, Japan.


   Abstract
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 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
The treatment of sepsis may require mechanical ventilation of the lungs and sedation. Because neutrophils are the most important effector cells for protecting against sepsis, and propofol and midazolam are the most widely used anesthetics for sedation, we studied the effects of these two anesthetics on the neutrophil function during sepsis. Sepsis was induced in rats by cecal ligation and puncture. At either 4 h or 24 h after cecal ligation and puncture, blood and peritoneal neutrophils were obtained, incubated with the test anesthetics, and the hydrogen peroxide (H2O2) production and CD11b/c expression were determined by flow cytometry. In both early (at 4 h) and late (at 24 h) sepsis, propofol and midazolam depressed H2O2 production by blood and peritoneal neutrophils at clinical concentrations. Propofol caused more depression than midazolam (P < 0.005). In both early and late sepsis, the effect of the anesthetics on the up-regulation of the stimulation-induced CD11b/c expression on blood neutrophils was minimal at clinical concentrations. If these results ultimately become clinically relevant, midazolam may be preferable to propofol for sedation during sepsis.

Implications: In septic patients, mechanical ventilation of the lungs is sometimes needed, and propofol and midazolam are widely used for sedation. Midazolam was less inhibitory for neutrophil function than propofol during sepsis; thus, midazolam may be preferable to propofol for sedation during sepsis for preserving the neutrophil function to combat sepsis.


   Introduction
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 Abstract
 Introduction
Methods
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 Discussion
 References
 
Sepsis is associated with increased morbidity and mortality (1,2). Because of the severity of the illness, sepsis patients sometimes need mechanical ventilation of the lungs. For mechanical ventilation, propofol and midazolam are probably the most widely used anesthetics for sedation (3).

Neutrophils are the major effector cells for protection against sepsis (4,5). Therefore, it may be desirable to use less inhibitory anesthetics during sepsis for preserving the neutrophil function to combat sepsis. Although the effects of propofol and midazolam on neutrophils have been investigated by using neutrophils from healthy volunteers (6–10), there are no reports studying, in a well-controlled manner, the effects of these drugs on neutrophils during sepsis. The behavior and properties of neutrophils in sepsis can be altered by several cytokines produced during sepsis (11–13) and thus neutrophils in sepsis may respond to these anesthetics differently from those of healthy subjects.

Sepsis cases are especially diverse: their concomitant illnesses, severity of sepsis, causative organisms of sepsis, and duration of sepsis may be greatly different from each other. Because these variables are difficult to control in clinical settings, we used the murine intraabdominal sepsis model (14). This model, in which sepsis was induced by cecal ligation and puncture (CLP), closely mimics the pathophysiology of sepsis in humans (15).

The aim of the study was to compare the effects of propofol and midazolam on neutrophil functions during sepsis. Neutrophils were obtained from rats undergoing sepsis, and the anesthetics were added to the neutrophils. The hydrogen peroxide (H2O2) production by neutrophils and the expression of CD11b/c, the major adhesion molecule on neutrophils (13,16), were measured for assessing neutrophil functions.


   Methods
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 Methods
Results
 Discussion
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After approval by the Institutional Committee on Animal Research, male specific pathogen-free Sprague-Dawley rats (weight 300–350 g; Charles River, Yokohama, Japan) were used for the experiments. Rats were maintained on a standard laboratory diet and housed in a controlled environment with a 12-h light/dark cycle.

We used a rat abdominal sepsis model (14) as follows. Rats (n = 26) were allowed access only to water for 6–10 h before the surgical procedure. Under sevoflurane anesthesia, the cecum was exposed through an abdominal midline incision. The cecum was ligated immediately below the ileocecal valve without obstruction of the ileum or colon, four holes were made in the cecum by puncture with an 18-gauge needle (n = 13), and the cecum was gently squeezed to extrude feces to ensure patency of the holes. The abdominal incision was closed and the rats were resuscitated with 13 mL of normal saline subcutaneously. After CLP, the rats were allowed free access to food and water. Sham-operated rats (n = 13) underwent the same procedure except for ligation and puncture of the cecum. All surgical procedures were performed in sterile fashion.

At either 4 h (n = 6 for each group) or 24 h (n = 7 for each group) after CLP or after the sham operation, rats were anesthetized with sevoflurane and heparinized (20 U · mL-1) blood was obtained by percutaneous cardiac puncture. Heparin is an anticoagulant of choice for experiments where neutrophils are to be stimulated as in our study (17,18). Then the abdominal wall was opened and the abdominal cavity was filled and mixed with 20 mL of phosphate buffered saline (PBS) containing 5 mM glucose and 0.1% gelatin (PBSg). Fifteen mL of the lavage fluid was then retrieved. Blood leukocytes were counted using a Coulter counter (Celltac; Nihon Kohden, Tokyo, Japan). Differentials of blood leukocytes were determined by enumeration of cells in Giemsa-stained blood smear preparations.

Propofol at concentrations of 5 µg · mL-1 and 50 µg · mL-1, and midazolam at concentrations of 1.5 µg · mL-1 and 15 µg · mL-1 were tested. In both cases, the former concentrations were expected to be the maximum doses of clinically relevant plasma concentrations for sedation of the drugs (9,10,19).

Neutrophils were isolated from blood using dextran sedimentation. The remaining erythrocytes were lysed with ice-cold distilled water for 20 s (hypotonic shock) and cells (105 cells · mL-1) were suspended in PBSg. The viability of the leukocytes was >98%, as determined by trypan blue exclusion. The peritoneal lavage fluid was washed with PBSg, contaminating red blood cells were lysed by hypotonic shock, and the peritoneal cells were resuspended in PBSg (105 cells · mL-1). The percentage of neutrophils in these peritoneal cells was determined by differentiation in Giemsa-stained cytospin preparations. Viability of the peritoneal cells was >98%, as determined by trypan blue exclusion. Autologous plasma was obtained by centrifugation (12,000g, 3 min) of the heparinized blood. Heat-killed (60°C for 30 min) Staphylococcus aureus (strain ATCC 25923, 2.4 x 109 colony forming units · mL-1) (supplied from Shionogi Pharmaceutical Co., Osaka, Japan) was incubated with an equal volume of the autologous plasma for 30 min at 37°C for opsonization, washed, and resuspended in PBSg (20).

Blood or peritoneal neutrophils (200 µL) were incubated with or without the anesthetics in PBSg (800 µL) for 5 min at 37°C. Then, 200 µL of 1.5 mM 2',7'-dichloro-fluorescin diacetate (DCFH-DA) (20,21) (Eastman Kodak, Rochester, NY) in PBSg was added and the mixture was incubated for 5 min at 37°C. The neutrophils were stimulated with 200 µL of the opsonized S. aureus at 37°C for 20 min. Stimulation was terminated by adding 1 mL of cold PBSg containing 3 mM ethylene diamine tetraacetic acid (Wako Chemicals, Tokyo, Japan). The cells were centrifuged, resuspended in 500 µL PBSg, and subjected to flow cytometry. In this assay, the stable nonfluorescent compound, DCFH-DA, diffuses into neutrophils and is hydrolyzed to 2',7'-dichlorofluorescin (DCFH), which is oxidized to fluorescent 2',7'-dichlorofluorescein (DCF). The intensity of fluorescence of DCF correlates with H2O2 production (21).

Heparinized blood (100 µL, aliquots) was incubated with or without anesthetics for 30 min at 37°C. Then, neutrophils were incubated with or without N-formyl-methionyl-leucyl-phenylalanine (fMLP) (10-6 M) (Sigma Chemical, St. Louis, MO) for 20 min at 37°C. Stimulation was terminated by adding 2 mL of cold PBS containing 2% fetal calf serum. The blood cells were washed and resuspended in 100 µL of PBS containing 2% fetal calf serum. Blood cells were incubated with 0.5 µg of fluorescein isothiocyanate-conjugated anti-CD11b/c antibody MRC OX-42 (Caltag, Burlingame, CA) at 4°C in the dark for 30 min. Fluorescein isothiocyanate-conjugated isotype control antibody (Caltag, Burlingame, CA) was used to detect nonspecific binding. The cells were washed with cold PBS containing 2% fetal calf serum, and red blood cells were lysed by a lysing solution containing 0.876% NH4Cl. The cells were centrifuged, resuspended in 500 µL PBSg, and subjected to flow cytometry. This whole blood staining technique is a sensitive and suitable way of assessing the neutrophil surface adhesion molecule expression at the time of blood collection (5,22).

An EPICS XL flow cytometer (Coulter) was used with a 488-nm argon laser. The fluorescence from fluorescein isothiocyanate and DCF was measured through a 530-nm filter. The flow cytometer was calibrated using fluorescent microspheres (Polyscience, Washington, PA) before each study to ensure standard laser intensity and alignment. Neutrophils were selected and gated by the typical forward and side light scatter profiles. Ten thousand cells were analyzed from each sample. The H2O2 production and the CD11b/c expression were reported as the mean fluorescence intensity.

Results are expressed as mean ± SEM. Comparisons were made by the factorial or two-way repeated measures of analysis of variance with Bonferroni correction. P < 0.05 was considered to be statistically significant.


   Results
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 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
The percentage of neutrophils in peritoneal cells was 94 ± 3% and 60 ± 6% at 4 h and 24 h after CLP, respectively, and 73 ± 8% and 40 ± 7% at 4 h and 24 h after the sham operation, respectively. Sepsis increased blood neutrophils (P < 0.005) and H2O2 production per cell in both blood (P < 0.05) and peritoneal (P < 0.005) neutrophils at 4 h (early sepsis) but not 24 h (late sepsis). The resting and fMLP-induced CD11b/c expression without anesthetics was not affected by sepsis ( Table 1).


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  		Table 1. Mean ± sem Blood Neutrophil Counts, Hydrogen Peroxide (H2O2) Production by Blood and Peritoneal Neutrophils (Mean Fluorescence Intensity), and Resting and N-formyl-methionyl-leucyl-phenylalanine (fMLP)-Induced CD11b/c Expression on Neutrophils (Mean Fluorescence Intensity)
 
In sepsis, both propofol and midazolam decreased the H2O2 production by the neutrophils both at 4 h and 24 h (P < 0.05) ( Fig. 1 (A), (B)). Propofol always caused a larger depression than midazolam (P < 0.005) both at clinical and 10-fold clinical concentrations. In sepsis, propofol and midazolam had a minimal effect on the fMLP-induced up-regulation of CD11b/c expression at clinical concentrations (Fig. 1 C). At 10-fold clinical concentrations, propofol and midazolam suppressed the up-regulation of CD11b/c expression both at 4 h and 24 h (P < 0.05). Midazolam suppressed the expression of CD11b/c more than propofol only at 10-fold clinical concentrations (P < 0.05).



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  		Figure 1. Effects of propofol and midazolam on the hydrogen peroxide (H2O2) production by blood (A) and peritoneal (B) neutrophils and the N-formyl-methionyl-leucyl-phenylalanine-induced CD11b/c expression on blood neutrophils (C), after CLP or sham operation. Data (mean ± SEM) are expressed as a percentage of the control (in the absence of IV anesthetics). x1: clinical concentrations (propofol: 5 µg · mL-1; midazolam: 1.5 µg · mL-1). x10: 10-fold clinical concentrations. CLP: cecal ligation and puncture. n = 6–7 for each group. *P < 0.05, **P < 0.005 between propofol and midazolam (compared only for sepsis group). {dagger}P < 0.05, {dagger}{dagger}P < 0.005 versus control [without anesthetics (0)] (compared only for sepsis group). #P < 0.05, ##P < 0.005 between sepsis and sham operation groups (compared by two-way repeated-measures analysis of variance).

 
At 24 h, blood neutrophils from sepsis were significantly more susceptible to the depressive effect of propofol on H2O2 production than those from sham-operated rats (P < 0.005) (Fig. 1 A). At 4 h, neutrophils from sepsis were significantly more resistant to the suppressive effect of midazolam on the fMLP-induced up-regulation of CD11b/c expression than those from the sham-operated rats (P < 0.05) (Fig. 1 C). Otherwise, the effects of anesthetics on neutrophils were not different between sepsis and sham operation groups.

The effect of the propofol carrier, 10% Intralipid, on H2O2 production by neutrophils is controversial (7,8). Therefore, we measured H2O2 production in the presence of Intralipid (Otsuka, Osaka, Japan) using blood obtained from normal rats (n = 6) and the procedure described in Methods. Intralipid dose-dependently depressed the H2O2 production (P < 0.05). The H2O2 production, expressed as a percentage of the control value without Intralipid, was 95 ± 3% and 91 ± 2% at concentrations of Intralipid corresponding to propofol concentrations of 5 µg · mL-1 and 50 µg · mL-1, respectively. Thus, the contribution of Intralipid was negligible.


   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
We found that propofol significantly inhibits H2O2 production more than midazolam at clinical concentrations during sepsis, whereas the effect of both anesthetics on the fMLP-induced up-regulation of the adhesion molecule, CD11b/c, was minimal at these concentrations. We also found that the effects of anesthetics on neutrophils were generally similar with or without sepsis.

At 4 h after CLP and without anesthetics, the H2O2 production by blood and peritoneal neutrophils was significantly more than that after sham operation, which means that neutrophils from early sepsis (at 4 h) are primed to produce a larger amount of H2O2 on stimulation. The fact that compared with that in sham operation, neutrophils produce a significantly larger amount of H2O2 in early sepsis but not in late sepsis (at 24 h) is consistent with the previous study (5). However, the resting CD11b/c expression without anesthetics was not different between sepsis and sham-operated rats. Because we used whole blood to stain the surface molecule, the cytokine milieu of sepsis could have been retained at the time of incubation. Therefore, we believe that the results reflect the actual level of CD11b/c expression at the time of the neutrophil harvest. A previous study (5) showed that the CD11b/c expression was significantly up-regulated at 6 h but not 24 h after CLP compared with the sham operation group. The reason for the lack of difference in the CD11b/c expression at 4 h is not clear, but the differences in time of sampling (at 4 h after CLP in our study versus 6 h in their study), in the severity of sepsis of the model (four cecum punctures versus two), and/or in the blood sampling technique (through direct cardiac puncture versus through abdominal aorta) may contribute to it.

The effects of the anesthetics on sepsis-primed neutrophils and nonprimed neutrophils were generally similar. We think that this is because the effects of the anesthetics on both sepsis-primed neutrophils and nonprimed neutrophils were more than the difference caused by the sepsis-priming effect alone, thereby resulting in a similar depression in both sepsis and sham operation. However, neutrophils from sepsis did respond to propofol and midazolam somewhat differently from those obtained from the sham-operated rats. Blood neutrophils from the early sepsis were more resistant to the suppressive effect of midazolam on the fMLP-induced CD11b/c expression, whereas those from the late sepsis were more susceptible to the depressive effect of propofol on H2O2 production than those in the sham-operated rats. This may be partly because of the production of proinflammatory cytokines in the early period of sepsis and the production of antiinflammatory cytokines, which inhibit the synthesis and release of the proinflammatory cytokines, in the late period of sepsis (13,23).

We used sevoflurane for all the invasive procedures. Because the effects of sevoflurane on neutrophils are minimal and dissipate within 30 min after its withdrawal (24), we assume that the use of sevoflurane minimally affected the results of our study.

Neutrophils were obtained from septic rats and anesthetics were added in vitro. We had the opinion that this was the easiest way to elucidate the effects of anesthetics on the sepsis-primed neutrophils. However, anesthetics may affect cells other than neutrophils, such as monocytes, macrophages, or endothelial cells, and may modulate the cytokine milieu of sepsis or the expression of endothelial adhesion molecules during sepsis (25). Thus, the overall effects of anesthetics on septic animals are difficult to predict based on our in vitro study. An in vivo study in which septic animals are given anesthetics is needed.

In summary, selection of anesthetics for sedation for mechanical ventilation during sepsis may be crucial because inhibition of neutrophil function by the anesthetics, if present, may be deleterious for septic patients, spoiling their neutrophils’ capacity to combat sepsis. If our data can be extrapolated to the clinical setting, midazolam may be preferable to propofol for sedation during sepsis.


   References
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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Accepted for publication October 17, 2000.




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Lippincott, Williams & Wilkins Anesthesia & Analgesia® is published for the International Anesthesia Research Society® by Lippincott Williams & Wilkins and Stanford University Libraries' HighWire Press®. Copyright 2001 by the International Anesthesia Research Society. Online ISSN: 1526-7598   Print ISSN: 0003-2999 HighWire Press