Anesth Analg 2002;94:385-388
© 2002 International Anesthesia Research Society
CRITICAL CARE AND TRAUMA
The Effects of Intravenous Anesthetics and Lidocaine on Proliferation of Cultured Type II Pneumocytes and Lung Fibroblasts
Kahoru Nishina, MD,
Katsuya Mikawa, MD,
Osamu Morikawa, MD,
Hidefumi Obara, MD, and
R. J. Mason, MD*
Department of Anesthesia and Perioperative Medicine, Faculty of Medical Sciences, Kobe University Graduate School of Medicine, Kobe, Japan; and *Department of Medicine, National Jewish Medical and Research Center, Denver, Colorado
Address correspondence and reprint requests to Katsuya Mikawa, MD, Department of Anesthesia and Perioperative Medicine, Faculty of Medical Sciences, Kobe University Graduate School of Medicine, Kusunoki-cho 7, Chuo-ku, Kobe 650-0017, Japan. Address e-mail to katz{at}med.kobe-u.ac.jp
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Abstract
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Type II pneumocytes synthesize surfactant and differentiate into type I pneumocytes to maintain the epithelium (1). Alveolar type II cell proliferation is required for reepithelization after acute lung injury (ALI) and is thought to minimize the subsequent fibrotic response (1). Keratinocyte growth factor (KGF) and hepatocyte growth factor (HGF) are among the most potent mitogen for type II epithelial cells, but not for fibroblasts in the lung (1). These growth factors attenuate several experimental ALI models by promoting epithelial repair (2,3). Thus, KGF and HGF may be a promising therapeutic approach to ALI. Critically ill patients with ALI often receive IV anesthetics or sedatives to facilitate mechanical ventilation. Furthermore, these patients sometimes undergo bronchoscopy under local anesthesia to obtain bronchoalveolar lavage fluid or to remove respiratory secretions. Several IV and local anesthetics inhibit pro-liferation of various cells including epithelium (4,5). If these anesthetics impede proliferation of type II pneumocytes, this suppressive effect may be a disadvantage for alveolar reepithelization in the course of recovery from ALI. In this study, we examined the effects of midazolam, propofol, ketamine, thiopental, and lidocaine on proliferation of type II alveolar epithelial cells using in vitro culture system. Because fibroblast proliferation is a key event in late phase of ALI, inhibition of this fibroproliferation is probably beneficial. Thus, we further determined whether these anesthetics could regulate proliferation of lung fibroblasts. In the current study, rolipram was used as a positive control. In our previous preliminary experiment, we found that rolipram, a phosphodiesterase inhibitor type IV, augments spontaneous or KGF-/HGF-promoted type II cell proliferation (6).
IMPLICATIONS: Midazolam, ketamine, thiopental, propofol, or lidocaine did not inhibit proliferation of cultured rat type II pneumocytes. Our findings suggest that these anesthetics do not impede alveolar reepithelization after acute lung injury.
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Methods
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The current study was approved by the animal care review board of Kobe University School of Medicine.
Type II cells were dissociated from the lungs of adult male Sprague-Dawley rats (200–250 g) as previously described (7). Briefly, perfused lungs were excised, lavaged, and then incubated with a fluoro-carbon-albumin emulsion. The lungs were lavaged again, filled with porcine pancreatic elastase solution, and incubated for 20 min at 37°C. The tissue was minced, and the cells were filtered and separated on discontinuous metrizamide density gradients. The type II cells from the gradient were resuspended in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum, supplemented with glutamine, penicillin, streptomycin, amphotericin B, and gentamicin sulfate. The purity of type II pneumocytes preparation assessed by modified Papanicolaou stain was 
90%. The cell viability assessed by erythrosin B exclusion test was 95%. The type II cells (5 x 104 cells/well in 100 µL of the medium) were plated into 96-well plastic dishes, and maintained at 37°C in a humidified incubator containing 95% air/5% CO2. The cells were allowed to adhere for 24 h, and rinsed with Dulbecco’s modified Eagle’s medium to remove nonadherent cells (37% plating efficiency in preliminary experiment). The cells were then incubated for another 48 h in the same medium containing various combinations of the growth factors (KGF, 1 and 10 ng/mL; HGF, 2 and 20 ng/mL) (8–10) and the anesthetics listed in Table 1. Midazolam, ketamine, thiopental, and lidocaine were diluted with distilled water. Propofol and rolipram were diluted with dimethylsulfoxide. Each anesthetic or rolipram was added to give final concentrations corresponding to 1, 10, and 100 times plasma concentrations observed clinically.
After the subsequent culture for 48 h, cell proliferation was assessed using WST-8 (modified tetrazolium salt) cell proliferation kit (Cell Counting Kit-8, Dojin, Japan) and bromodeoxyuridine (BrdU) incorporation kit (Cell Proliferation ELISA, BrdU (colorimetric) Kit; Roche, Basel, Switzerland) according to the manufacturers’ protocols instead of direct cell count. The former and the latter assays reflected metabolic activity and DNA synthesis, respectively. In a preliminary study, we confirmed that there is a good correlation between optical densities obtained by the kits and cell number counted using a hemocytometer in type II cells. These kits also provide timesaving procedures and have high sensitivity and specificity. In a subsequent experiment, adult human lung fibroblasts (HLF) (WI38, Riken, Japan; 5 x 104 cells/well in 100 µL of the medium) were used instead of type II pneumocytes. Data were analyzed for statistical significance by Friedman’s rank test followed by Dunnett’s test for post hoc comparisons. P < 0.05 was deemed significant.
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Results
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KGF and HGF enhanced cellular metabolic activity and DNA synthesis suggestive of cell proliferation (Table 1). The five anesthetics failed to change spontaneous or KGF/HGF-induced proliferation of type II cells as assessed by WST-8 assay (Table 1). The anesthetics did not affect HLF proliferation regardless of the presence of the growth factors, except that lidocaine 200 µg/mL inhibited HLF proliferation (Table 2). The effects of the anesthetics determined by the BrdU assay were similar (data not shown).
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Discussion
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Important functions of alveolar type II cells include synthesis and secretion of surfactant, maintenance of the alveolar epithelium by their ability to proliferate and differentiate into type I cell, and transepithelial transport of sodium from the apical to the basolateral surface to minimize alveolar fluid (1). Restoration of the alveolar epithelium in ALI occurs by proliferation of type II cells. In the current study, we have confirmed that KGF and HGF promoted proliferation of type II pneumocytes. There was agreement on experimental results between the two different cell-counting methods with high specificity based on metabolic activity or DNA synthesis. This consistency would increase reliability of our results to minimize false-positive results. We first found that midazolam, ketamine, thiopental, propofol, or lidocaine did not influence proliferation of type II pneumocytes. Our findings suggest that these IV/local anesthetics do not impede alveolar reepithelization after lung damage. Inhibition of lung fibroblasts’ proliferation is probably advantageous after lung injury. In the current study, we also found that these anesthetics failed to attenuate HLF proliferation, except that large concentrations of lidocaine successfully inhibited it. This lidocaine-induced inhibition may be a result of membrane depolarization (11). However, the beneficial effect of lidocaine is unlikely to be feasible in clinical settings because the drug, at clinically relevant concentrations, failed to reduce HLF proliferation.
Cyclic AMP increasing agents intensify proliferation of various epithelial cells (12,13). In the current study, we also confirmed that rolipram increased spontaneous and KGF/HGF-promoted proliferation of type II pneumocytes, although the mechanism remains unresolved. Rolipram attenuates experimental ALI models (14,15). The successful application of the drug may be attributable to its ability to enhance proliferation of type II cells but not fibroblasts. Rolipram may be a possible alternative to KGF or HGF in therapeutic modality for ALI, and concomitant use of the growth factors and rolipram may provide a therapeutic approach more promising than that provided by either agent alone.
In the current study, we preliminarily determined only the proliferative function of the pneumocytes contributing to alveolar reepithelization. Exposure to thiopental increases expression of surfactant associated protein-C mRNA in cultured rat alveolar type II cells (16). Further studies are required to determine whether the anesthetics tested in the current study can alter surfactant synthesis and alveolar sodium transportation. The in vivo effect of these anesthetics on proliferation of lung cells in animals also deserves further study.
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References
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Accepted for publication September 20, 2001.
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Abstract
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