Previous studies indicate that anesthesia and surgery induce an inflammatory reaction in alveolar macro phages. However,they filed to independently evaluate the relative contributions of factors including mechanical ventilation, general anesthesia, and surgical stress. Therefore, the authors tested the hypothesis that inflammatory reactions at the cellular level in alveolar macrophages are induced within 2 h of inhalation of volatile anesthetics under mechanical ventilation.
After administration of pentobarbital, rats were allocated to the nonventilated control or spontaneous or mechanical ventilation (n = 15/group) for 2 h at a fraction of inspired oxygen (FI(O2)) of 0.21. In a separate series of experiments, rats were mechanically ventilated without volatile anesthesia, or during exposure to halothane, enflurane, isoflurane, or sevoflurane (n = 15/group). Pulmonary lavage was performed, and RNA was extracted from harvested cells. The mRNA for the proinflammatory cytokines interleukin (IL)-1alpha, IL-1beta, IL-6, macrophage inflammatory protein-2 (MIP-2), interferon gamma (IFN-gamma), and tumor necrosis factor alpha (TNF-alpha) were measured by semiquantitative reverse transcription-polymerase chain reaction using beta-actin as an internal standard. Pulmonary lavage concentrations of these cytokines were measured by enzyme-linked immunoassay.
The lavage cell count and cytology were similar in each series of the experiment. Gene expression of MIP-2 and TNF-alpha was greater during mechanical than spontaneous ventilation and nonventilation control However, the concentrations of cytokines except MIP-2 and TNF-alpha were less than detection levels. During exposure to volatile anesthetics, gene expression for IL-1beta, MIP-2, IFN-gamma, and TNF-alpha all increased significantly compared with mechanical ventilation alone. Significant increases in lavage concentrations of MIP-2 and TNF-alpha were also observed.
Gene expression of proinflammatory cytokines increase after inhalation of volatile anesthetics under mechanical ventilation. These data indicate that inhalation of volatile anesthetics under mechanical ventilation induces an inflammatory response at the transcriptional level within 2 h.
ALVEOLAR macrophages are the first line of pulmonary defense. In previous studies, we reported that an influx of neutrophils and alveolar macrophage aggregation increased as the duration of anesthesia and surgery increased. [1,2] Macrophage aggregation and influx of neutrophils are induced by various immunomodulators and characterize inflammatory reactions in the lungs. [3–5] Interestingly, these responses were minimal within 2 h of anesthesia and surgery. Although neutrophil influx and alveolar macrophage aggregation were minimal within 2 h of anesthesia and surgery, it is likely that an inflammatory reaction is induced at cellular levels in alveolar macrophages, such as gene expression and production of proinflammatory cytokines.
Our previous human studies did not independently evaluate the relative contributions of factors including mechanical ventilation, general anesthesia, and surgical stress. We believe that each of these factors may contribute significantly to observed intraoperative pulmonary inflammatory responses. Surgical stress obviously induces a generalized inflammatory response.  The effects of volatile anesthetic inhalation during mechanical ventilation is also of interest, because our previous study showed that macrophage aggregation is more pronounced during isoflurane than propofol anesthesia.  Finally, it is well established that mechanical ventilation can augment inflammatory reactions in the injured lungs. 
To evaluate the relative contribution of anesthesia to the inflammatory response of alveolar macrophages, we tested the hypothesis that inflammatory reactions at cellular levels of alveolar macrophages are induced within 2 h of inhalation of volatile anesthetic under mechanical ventilation. In addition to measuring concentrations of proinflammatory cytokines in pulmonary lavage fluid, we used the reverse transcription polymerize chain reaction (PCR) to amplify mRNA of proinflammatory cytokines and detect changes in alveolar macrophages at the transcriptional level. Given the complexity and redundancy of the cytokine network, [8,9] we evaluated the key proinflammatory cytokines, including interleukin (IL)-1 [Greek small letter alpha], IL-1 [Greek small letter beta], IL-6, macrophage inflammatory protein-2 (MIP-2), interferon gamma (IFN-[Greek small letter gamma]), and tumor necrosis factor alpha (TNF-[Greek small letter alpha]).
The study protocol was approved by the Institutional Animal Care Committee at the University of Hirosaki. One hundred twenty Wistar male rats weighing approximately 250 g were studied. All rats were maintained under standard conditions with free access to water and rodent laboratory food. Artificial light was present from 7 A.M. to 7 P.M. each day, and room temperature was maintained between 22 [degree sign]C and 24 [degree sign]C. The experiments in each group for each study were performed at randomly selected times ranging from 9 A.M. to 5 P.M.
After the animals were anesthetized with 50 mg/kg intraperitoneal pentobarbital, the trachea was excised and a catheter (Angiocath, 16 gauge; Deseret Medical, Sandy, UT) was inserted into the trachea, Catheters (Angiocath, 24 gauge) were also inserted into the femoral artery and vein. Lactated Ringer's solution was infused at a rate of 10 ml [middle dot] kg-1[middle dot] h-1through the venous catheter. We continuously monitored mean arterial pressure and heart rate from the femoral arterial catheter. The rats were placed on a heating pad (Small Animal Warmer BWT-100; BRC Co., Nagoya, Japan) and rectal temperature was maintained between 37 [degree sign]C and 37.5 [degree sign]C.
Experimental Protocol: Ventilation Modes
The purpose of this experiment was to evaluate gene expression and production of proinflammatory cytokines after spontaneous and mechanical ventilation. Forty-five rats were equally assigned to one of three groups: The control group underwent neither spontaneous nor mechanical ventilation. That is, they were killed for pulmonary lavage (n = 10) and to determine wet-to-dry ratios (n = 5) immediately after induction of pentobarbital anesthesia and tracheostomy. A second group of animals (n = 15) was mechanically ventilated for 2 h, whereas rats in the third group (n = 15) breathed spontaneously during this period.
The mechanically ventilated rats were paralyzed by a continuous infusion of 0.1 ml [middle dot] kg-1[middle dot] h-1vecuronium. A rodent ventilator (model 683; Harvard Apparatus, South Natick, MA) set to 10 ml/kg was used in the mechanically ventilated animals. Arterial blood was sampled for analysis of pH, oxygen tension, and carbon dioxide tension at the beginning of each experiment (initial values) and again 2 h later (final values) except in the control group. The respiratory rate was controlled to produce an initial arterial carbon dioxide tension between 35 and 45 mmHg. Two hours after the start of the experiments, 10 rats in the ventilated groups were killed for whole pulmonary lavage, and the remaining five rats were used to evaluate the wet-to-dry weight ratios and histopathologic changes that occurred in the lungs.
Experimental Protocol: Volatile Anesthetic Exposure
The purpose of the experiment was to evaluate the effect of volatile anesthetic on alveolar macrophages in mechanically ventilated rats. We assigned 75 rats to one of five groups of 15 animals: One group was mechanically ventilated with continuous infusion of 0.1 mg [middle dot] kg (-1)[middle dot] h-1vecuronium. The remaining animals were mechanically ventilated with 1.5 minimum alveolar concentration halothane (1.6%), enflurane (3.4%), isoflurane (2.1%), or sevoflurane (3.7%) supplemented with continuous infusion of 0.1 mg [middle dot] kg-1[middle dot] h-1vecuronium. The rodent ventilator was set as described before in the ventilation modes protocol. Arterial blood was sampled for analysis of pH, oxygen tension, and carbon dioxide tension immediately after the start of the experiment in the control group or immediately after the start of inhalation of volatile anesthetics in the remaining four groups (initial values) and again 2 h later (final values). Ten and five rats in each group were used for whole pulmonary lavage and histopathologic examination of the lungs, respectively.
Histopathologic Examination and the Wet-to-dry Ratios
Immediately after the rats were killed, we measured pulmonary wet:dry ratios to evaluate pulmonary congestion. The right upper lobe was weighed and then dried to a constant weight at 60 [degree sign]C for 24 h.
The lower lobe of the right lung was fixed by the instillation of 10% formaldehyde solution through the right lower bronchus at a pressure of 20 cm water. The fixed lung was embedded and hematoxylin and eosin and Mallory-Azan stains were applied. We graded the degree of the lung injury using a five-point scale according to a combined assessment of alveolar and interstitial edema, hemorrhage, and accumulation and aggregation of immune cells based on a previously reported system: 0 = minimum damage, 1 = mild damage, 2 = moderate damage, 3 = severe damage, and 4 = maximum damage. 
Whole Pulmonary Lavage and Cell Isolation
Whole pulmonary lavage was performed in 10 rats from each group. Five milliliters saline solution, 0.9%, containing 16 mM lidocaine hydrochloride titrated with NaOH to a pH of 7.4 was instilled gently via a syringe and then withdrawn. This procedure was repeated 10 times, so that 50 ml of the saline solution was instilled. Three-milliliter aliquots of lavage fluid were reserved for total cell count, viability, and cell differentiation and aggregation. The rest was centrifuged immediately at 200g for 10 min. After the supernatant was decanted to measure cytokine concentrations, the cell pellets were dissolved in 0.5 ml of guanidinium buffer solution (4 M guanidinium isothiocyanate, 50 mM Tris HCl, 10 mM EDTA, 2% sarcoryl, and 100 mM mercaptoethanol).
The total cell number was determined using a hemocytometer. The viability of alveolar cells was evaluated by an ability to exclude the 0.2% trypan blue, as described in our previous studies. [1,2] Cell differentiation and aggregation were examined by counting 500 cells on a Wright-Giemsa-stained slide.
RNA Isolation and cDNA Synthesis
RNA was isolated from the guanidinium buffer by the well-established acid guanidinium-phenol-chloroform method.  The amount of isolated RNA was measured using a spectrophotometer (model DU-65; Beckman, Tokyo, Japan). We obtained 2.4–3.8 [micro sign]g RNA from each sample. By incubation at 40 [degree sign]C for 60 min, cDNA was synthesized from 2 [micro sign]g RNA with 20 [micro sign]l total reaction mixture, including Tris-HCl buffer (pH 8.3), 1 mM dNTPs, and 0.125 [micro sign]M oligo dT primers, and 20 U RNase inhibitor and 0.25 U AMV reverse transcriptase. After 60 min of incubation, the reverse transcriptase was inactivated at 95 [degree sign]C for 5 min.
The Semiquantitative Reverse Transcription Polymerase Chain Reaction
The reverse transcription polymerase chain reaction (PCR) mixture (50 [micro sign]l) contained cDNA synthesized from 0.2 [micro sign]g RNA, 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 2.5 mM MgCl2, 0.2 mM dNTP, 0.2 [micro sign]M 5′ and 3′ oligonucleotide primers, and 2.5 U Taq polymerase (Takara, Co., Tokyo, Japan).
The reaction mixture was amplified in a DNA thermocycler (Perkin-Elmer Co., Irvine, CA). Each cycle consisted of denaturation at 94 [degree sign]C for 1 min, annealing at 56 [degree sign]C (for IL-6 and IFN-[Greek small letter gamma]) or 59 [degree sign]C (for other cytokines) for 1 min, and extension at 72 [degree sign]C for 1 min. The optimal number of PCR cycles for each primer set was determined in preliminary experiments so that the amplification process was performed during the exponential phase of amplification.  The number of PCR cycles are as follows; 27 for [Greek small letter beta]-actin, 32 for IL-1 [Greek small letter alpha], 31 for IL-1 [Greek small letter beta], 35 for IL-6, 28 for IFN-[Greek small letter gamma], 33 for MIP-2, and 28 for TNF-[Greek small letter alpha]. The sequence of cytokine-specific primer pairs, 5′ and 3′, is shown in Table 1. Coamplification of the cDNA for each cytokine and [Greek small letter beta]-actin was performed in single tubes. The [Greek small letter beta]-actin primers were added after several cycles with only cytokine primer so that the final number of PCR cycles was optimal for both the cytokine and [Greek small letter beta]-actin.
The PCR products were quantified by densitometry measurements. The PCR products were separated by electrophoresis on a 1.8% agarose gel containing 0.5 [micro sign]g/ml ethidium bromide. The PCR products were visualized on a transilluminator (model FBTIV-816; Fisher Scientific, Pittsburgh, PA) at 312 nm wavelength and photographed with Polaroid 667 film (Japan Polaroid, Tokyo, Japan). The band images were obtained by scanning the image with a ScanJet 3P (Hewlett-Packard, Andover, MA). The total intensity (average intensity x total pixels) of each band was measured using Mocha software (Jandel Scientific Software, San Rafael, CA). To evaluate the relative amount of cytokine mRNA in each rat, the cytokine-to-[Greek small letter beta]-actin ratio of the intensity of ethidium bromide luminescence for each PCR product was calculated. 
The Cytokine Enzyme-linked Immunoassay in Whole Pulmonary Lavage Fluid
Cytokine analysis on the whole pulmonary lavage fluid (IL-1 [Greek small letter alpha], IL-1 [Greek small letter beta], IL-6, MIP-2, IFN-[Greek small letter gamma], TNF-[Greek small letter alpha]) was performed in duplicate in a blinded manner using commercially available enzyme-linked immunoassay kits (TFB Co., Tokyo, Japan). The minimum detection levels were 5, 5, 31, 1, 7, and 0.7 pg/ml, respectively. In our laboratory, the intra- and interassay coefficients of variation of each cytokine measurement are less than 5% and 8%, respectively. All kits except IL-1 [Greek small letter alpha] were specific for the rat. The kit for IL-1 [Greek small letter alpha] was a murine preparation having suitable cross-reactivity with rat IL-1 [Greek small letter alpha]. The absorbance of each well was read at 450 nm using a microplate reader. Background absorbency of blank wells was subtracted from the standard and unknowns before the sample concentration was determined.
We used nonparametric methods for statistical analyses. Wilcoxon signed-rank, Mann-Whitney U, and Kruskal-Wallis tests were used when appropriate. Data are expressed as mean +/- SD; P < 0.05 was considered significant.
(Table 2) shows changes in cardiorespiratory parameters in the ventilation modes protocol. The mean arterial pressure and heart rate did not change over time in the spontaneous and mechanical ventilation groups. Although the inhaled oxygen fraction was only 0.21, the arterial oxygen pressure was more than 80 mmHg in all animals; arterial oxygen pressure and pH also did not change over time or among groups. Although the arterial carbon dioxide tension decreased slightly at the final time point in animals in the spontaneous ventilation group, differences between spontaneous and mechanical ventilation were not observed. There were no differences in recovery rate, total cell number, cell distribution, aggregation, viability or wet-to-dry ratios among the three groups. Histologic changes in the lungs were minimal in all the rats studied (Table 3).
The relative concentrations of mRNA for MIP-2 and TNF-[Greek small letter alpha] in the pulmonary lavage fluid were significantly greater in the animals undergoing mechanical ventilation than in those without ventilation (P < 0.05), whereas there was no significant difference between the nonventilated controls and the spontaneous ventilation group (Figure 1). The concentration of IL-1 [Greek small letter alpha], IL-1 [Greek small letter beta], IL-6, and IFN-[Greek small letter gamma] were less than the detection level in pulmonary lavage fluid. None of the other pulmonary lavage cytokine concentrations differed among the three groups (Figure 2).
Volatile Anesthetic Exposure
(Table 4) shows the changes in cardiorespiratory parameters in each group. Both in the initial and final time points, mean arterial pressure and heart rate were significantly less in rats exposed to volatile anesthetics than in the control rats that were mechanically ventilated without volatile anesthesia. Core temperature, pH, respiratory rate, and the partial pressures of oxygen and carbon dioxide in arterial blood did not differ significantly with time or among groups. There were no differences in cell recovery rate, total cell number, cell distribution, aggregation, or viability among the groups. In all animals, histologic changes were minimal and did not include edema, hemorrhage, or infiltration of immune cells into alveolar spaces. We also failed to observe differences in the wet-to-dry ratios among the groups (Table 5).
The relative amount of mRNA for IL-1 [Greek small letter beta] and MIP-2 increased significantly in all groups with volatile anesthetics compared with the control group that was not given volatile anesthesia. The relative amount of mRNA for TNF-[Greek small letter alpha] was significantly greater than control in the rats given halothane, enflurane, or isoflurane. Furthermore, IFN-[Greek small letter gamma] mRNA increased significantly in the groups given halothane or enflurane (Figure 3).
(Figure 4) shows the cytokine concentrations in the pulmonary lavage fluid. As in the ventilation modes protocol, IL-1 [Greek small letter alpha] and IL-6 were undetectable. The level of MIP-2 in pulmonary lavage fluid was significantly greater in the groups given halothane, enflurane, and isoflurane than in those without volatile anesthesia. The concentrations of TNF-[Greek small letter alpha] in the lavage fluid were also significantly greater in the groups given halothane or enflurane than in those without volatile anesthetics. The concentrations of IL-1 [Greek small letter beta] and IFN-[Greek small letter gamma] in the pulmonary lavage fluid did not differ significantly among the groups (Figure 4).
Mechanical ventilation induced a slight increase in gene expression of the proinflammatory cytokines MIP-2 and TNF-[Greek small letter alpha]. In our study, carbon dioxide tension and respiratory rate did not differ between spontaneous and mechanical ventilation. The contributions of pentobarbital and tracheostomy to this response were minimal, because there were no differences in message pattern or cytokine production between spontaneous ventilation and the control groups. Our results thus suggest that positive airway pressure is the most likely factor contributing to the observed increase in gene expression.
The marked increase in gene expression of TNF-[Greek small letter alpha] has been reported during mechanical and high-frequency ventilation in a surfactant-deficient rabbit model.  Our study showed that an increase in gene expression of proinflammatory cytokines also occurs in the normal uninjured lungs. Although the exact mechanism of the increase in MIP-2 gene expression cannot be determined from our data, one study showed that gene expression of MIP-2 increased faster than did gene expression for the other proinflammatory cytokines. 
The notable finding in this study is that expression of proinflammatory cytokines increased after 2 h of inhalation of volatile anesthetics during mechanical ventilation. It is well established that alveolar macrophages react mainly to inhaled substances. For example, gene expression for proinflammatory cytokines increases after inhalation of noxious substances such as aerosolized lipopolysaccharide or bacteria, [15–17] nitrogen dioxide,  ozone,  and even 100% oxygen.  Our results, together with previous studies, thus strongly suggest that an inflammatory reaction is induced at the transcriptional level within 2 h of exposure to volatile anesthetics under mechanical ventilation.
It is interesting that both the expression and concentration of MIP-2 increased simultaneously. MIP-2 is one of the most potent chemoattractants for neutrophils to the distal airway. For example, tracheal instillation of recombinant rat MIP-2 causes neutrophil accumulation 2 h after administration.  We did not observe an increased number of neutrophils in the pulmonary lavage fluid. This result is consistent with our previous study in which neutrophil influx was not observed until 4 h after anesthesia and surgery. Ulich et al.  observed neutrophil influx to the distal airway 4–6 h after administration of lipopolysaccharide.
We also observed increases in both the expression and concentration of TNF-[Greek small letter alpha] 2 h after inhalation of volatile anesthetics during mechanical ventilation. Although the mechanism cannot be determined from this study, TNF-[Greek small letter alpha] and IL-1 [Greek small letter beta] are the most important cytokines secreted from alveolar macrophages. Gene expression of TNF-[Greek small letter alpha] is reported to be rapid; for example, administration of lipopolysaccharide causes a marked increase in expression of the TNF-[Greek small letter alpha] gene within 30 min. Even the inhalation of 100% oxygen increases the gene expression of TNF-[Greek small letter alpha] within 3 h.  Consistent with these observations, the production of TNF-[Greek small letter alpha] cytokines is much faster in IL-1[Greek small letter beta]. [21,22]
We observed significant increases in gene expression for IL-1 [Greek small letter beta] and IFN-[Greek small letter gamma], both of which are potent proinflammatory cytokines. Although we observed significant increases in gene expression for these cytokines, the concentrations did not increase in alveolar lavage fluid. Obviously, the increases in gene expression do not necessarily mean that the protein increases. However, the most obvious reason for discrepancy is that the study was insufficiently long. For example, protein expression of TNF-[Greek small letter alpha] was not detected until 24 h after exposure to 100% oxygen.  Similarly, alveolar macrophages secreted TNF-[Greek small letter alpha] and IL-1 [Greek small letter beta] 4 and 8 h after lipopolysaccharide stimulation, respectively. [21,22] An additional possibility, however, is that cytokine production may be suppressed by volatile anesthetics. For example, Voisin et al.  and Hammer and Rannels  reported that exposure of alveolar macrophages to volatile anesthetics decreases protein production and intracellular ATP.
In this experiment, each volatile anesthetic produced a different pattern of gene expression and protein production. Many studies have shown suppression of neutrophil function with halothane and enflurane. [25–28] In contrast, isoflurane has only a minimal effect on the neutrophil function. [25,27] Mitsuhata et al.  reported that production of TNF-[Greek small letter alpha] and IL-1 [Greek small letter beta] is inhibited by enflurane, isoflurane, and sevoflurane. Sevoflurane was most effective at inhibiting cytokine release. This finding is consistent with our findings. To our knowledge, however, no studies have been published concerning the effects of various volatile anesthetics on gene expression or production of proinflammatory cytokines. Additional studies clearly are needed to elucidate the effects of various volatile anesthetics on alveolar macrophage function, and to determine why they provoke different responses. Our findings indicate that transcriptional regulation of cytokines is an important feature 2 h after the inhalation of volatile anesthetics under mechanical ventilation.
We could not detect gene expression and protein for IL-1 [Greek small letter alpha] and IL-6. Why this should be so remains unclear. IL-1 [Greek small letter alpha] accounts for more than 90% of the IL-1 mRNA.  The failure of IL-1 [Greek small letter alpha] detection may be attributed to the fact that the function of IL-1 [Greek small letter alpha] is mainly intracellular and that IL-1 [Greek small letter alpha] may be released by cellular damage.  Xing et al.  reported that expression of the gene for IL-6 was even more difficult to induce than of IL-1 [Greek small letter alpha], even with lipopolysaccharide. In contrast, von Bethmann et al.  reported that the gene expression of TNF-[Greek small letter alpha] and IL-6 were similar in positive- and negative-pressure ventilation on isolated mouse lungs. This discrepancy may be explained by the experimental model. One possibility for the failure of IL-6 detection may be the enzyme-linked immunoassay kit used in this study, because the minimum detection level (31 pg/ml) was much higher than for the other cytokines.
We did not separate the macrophages from other cells, such as lymphocyte and neutrophils, to avoid any artificial influence of the sorting process on cytokine expression. For example, even adherence to plastic test tubes for separation of alveolar macrophage can induce gene expression of cytokines.  Thus, we cannot draw a definite conclusion regarding the cell source for the observed increase in gene transcription. However, macrophages are the likely source, because there is considerable evidence that the produce most proinflammatory cytokines.  Furthermore, at least 95% of the harvested cells were macrophages.
In conclusion, we found that inhalation of volatile anesthetics under mechanical ventilation augments gene expression of proinflammatory cytokines. Furthermore, some of cytokine concentration increased after the experiment. Our results indicate that transcriptional regulation of cytokines is an important feature 2 h after inhalation of volatile anesthetics under mechanical ventilation.