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Desflurane Differentially Affects the Release of Proinflammatory Cytokines in Plasma and Bronchoalveolar Fluid of Endotoxemic Rats

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Desflurane Differentially Affects the Release of Proinflammatory Cytokines in Plasma and Bronchoalveolar Fluid of Endotoxemic Rats 1139-1144 2/5/06 15:10 Page 1139 INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE 17: 1139-1144, 2006 Desflurane differentially affects the release of proinflammatory cytokines in plasma and bronchoalveolar fluid of endotoxemic rats KIM A. BOOST1, CHRISTIAN HOFSTETTER1, MICHAEL FLONDOR1, CHRISTIAN BETZ1, MARKUS HOMANN1, JOSEF PFEILSCHIFTER2, HEIKO MUEHL2 and BERNHARD ZWISSLER1 1Department of Anaesthesiology, Intensive Care and Pain Therapy and 2Pharmazentrum Frankfurt/ZAFES, Johann Wolfgang Goethe-University, 60590 Frankfurt am Main, Germany Received December 8, 2005; Accepted January 30, 2006 Abstract. Previous studies have indicated that volatile impaired in the Desflurane-group as compared to the LPS-only anaesthetics can attenuate the inflammatory response to group. Our data implicate that inhalation of 1MAC Desflurane lipopolysaccharide (LPS) and other proinflammatory stimuli during experimental endotoxemia differentially affects the in vitro and in vivo. Thus far, no studies are available on the inflammatory response in rats. influences of desflurane on the cytokine-release. We therefore aimed to investigate the effects of desflurane on the systemic Introduction and pulmonary release of proinflammatory cytokines in endo- toxemic rats. Eighteen anaesthetized and ventilated Sprague- Inflammation caused by endotoxemia or traumatic injury Dawley rats were randomly assigned to the following groups: leads to a strong release of proinflammatory cytokines in LPS-only: Six animals received LPS (5 mg/kg, i.v.) with no bronchoalveolar fluid and plasma (1,2). Several previous further intervention. LPS-Desflurane: Six animals received studies have indicated that exposure to volatile anaesthetics, continuous inhalation of 1MAC Desflurane before and during such as isoflurane, enflurane and halothane, attenuates the endotoxemia with LPS (5 mg/kg, i.v.). Sham: Six animals inflammatory response through decreasing the cytokine served as control without inhalation of desflurane and endo- release from alveolar macrophages and monocytes following toxemia. After 4 h, levels of tumor necrosis factor-· (TNF-·), stimulation with lipopolysaccharide (LPS) (3-5). Moreover, interleukin-1ß (IL-1ß) and interleukin-6 (IL-6) in plasma and volatile anaesthetics modulate the immune response by bronchoalveolar fluid were analyzed. Nitrite production as a decreasing neutrophil functions in vitro (6) and cytokine readout for nitric oxide (NO) release from alveolar macro- release by peripheral mononuclear cells (7). More recently, we phages was measured by Griess assay. IκB-· degradation and described that even a brief inhalation of isoflurane (50 sec) iNOS-protein in macrophage homogenates were determined markedly decreased the levels of proinflammatory cytokines by Western Blotting. Inhalation of desflurane during in plasma of endotoxemic rats (8). endotoxemia showed a significant decrease in release of the Among the inhalational anaesthetics little is known about proinflammatory cytokines TNF-· (-61%, P≤0.05) and IL-1ß the influence of desflurane on the inflammatory response (-47%, P≤0.05) in plasma as compared to LPS-only group, of alveolar macrophages and mononuclear cells. To our whereas the release of IL-6 was not significantly affected by knowledge, there are no studies concerning the impact of desflurane. Within the lung, the NO-release was notably desflurane on the cytokine release during inflammation. increased in supernatants of cultured alveolar macrophages Because of the growing use of desflurane in clinical practice, from desflurane-group compared to both LPS-only and the evaluation of possible immunomodulative properties is Sham group. IκB-· degradation in alveolar macrophages was of sustained interest for patients with systemic inflammatory response syndrome (SIRS) or acute lung injury/ARDS. Therefore, the aim of this study was to examine the effects _________________________________________ of desflurane on the inflammatory response indicated by the release of proinflammatory cytokines in plasma and broncho- Correspondence to: Dr Kim Alexander Boost, Department of alveolar fluid during experimental endotoxemia with LPS. Anaesthesiology, Intensive Care and Pain Therapy, Klinikum der Johann Wolfgang Goethe-Universität, Frankfurt am Main, Theodor- Materials and methods Stern-Kai 7, 60590 Frankfurt am Main, Germany E-mail: [email protected] Animals and anaesthesia. All animal experiments were approved by the governmental board for the care of animal Key words: desflurane, rat, lipopolysaccharide, interleukin-1ß, subjects (Regierungspräsidium Darmstadt, Germany) in tumor necrosis factor-· accordance to the Declaration of Helsinki and received care in compliance with the ‘Guide for the Care and Use of 1139-1144 2/5/06 15:10 Page 1140 1140 BOOST et al: DESFLURANE AND CYTOKINE LEVELS IN ENDOTOXEMIA Table I. Heart rate, mean arterial blood pressure and BALF cell count. ––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– Time Baseline 1 h 2 h 3 h 4 h ––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– HR [1/min] Sham 338 (26) 333 (27) 333 (34) 327 (18) 320 (18) LPS-only 356 (44) 348 (55) 354 (50) 364 (24) 372 (43) Desflurane 352 (39) 330 (39) 344 (29) 364 (23) 362 (31) MAP [mmHg] Sham 115 (35) 105 (14) 97 (23) 97 (23) 96 (24) LPS-only 103 (28) 91 (28) 89 (29) 90 (22) 98 (7) Desflurane 129 (30) 101 (26) 91 (24) 109 (20) 109 (13) TCC-BALF[x106/ml] Sham 3.8 (0.9) LPS-only 2.8 (0.4)a Desflurane 1.9 (0.2)b ––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– TCC-BALF = total cell count - bronchoalveolar lavage fluid. Each value represents mean (± SD), Kruskal-Wallis one way analysis of variance on ranks, Student-Newman-Keuls test. aLPS-only vs. Sham, bDesflurane vs. Sham, P<0.05. ––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– Laboratory Animals’, National Academic Press, Washington concentration (MAC) (6%) and fentanyl (2.5-5 μg/kg/h, i.v.). D.C. 1996. Male Sprague-Dawley rats (n=18) were obtained Concentration of desflurane was measured continuously by from Harlan Winkelmann (Borchen, Germany; mean body using a monitor for volatile anaesthetics (Vamos, Draeger weight ± SEM, 490±30 g) and kept on a 12-h light/dark cycle Medical, Lübeck, Germany). Thereafter, all animals except the with free access to food and water. Rats were anaesthetized Sham-group received i.v. endotoxin (lipopolysaccharide, LPS, initially by i.p. injection of pentobarbital (Narcoren, Merial, E. coli (055:B5), Sigma, Deisenhofen, Germany, 5 mg/kg). Halbergmoos, Germany; 50 mg/kg) plus fentanyl (Janssen- After 4 h of endotoxemia, animals were exsanguinated and Cilag, Neuss, Germany; 0.05 mg/kg). Unconscious rats were plasma samples were obtained. tested for sufficient depth of anaesthesia by tail clamping, weighed and then placed supine on a heating pad. A tracheo- Bronchoalveolar lavage (BAL). After thoracotomy, a broncho- tomy was immediately performed and a 13 G cannula (ID: alveolar lavage (BAL) was performed with 10 aliquots of 10 ml 2.0 mm, OD: 2.5 mm, Abbott, Wiesbaden, Germany), modified sterile, nonpyrogenic phosphate-buffered saline (PBS; Serva, with a standard connector for pediatric endotracheal tubes, was Heidelberg, Germany). Every rinse with a 10 ml aliquot was endotracheally inserted. Subsequently, rats were ventilated with performed standardized in 30 sec. The first aliquot of BAL- an infant ventilator (Stephanie®, Stephan, Gackenbach, fluid samples was separated from the following aliquots and Germany) using pressure controlled ventilation: pmax 1.6 kPa, centrifuged. The supernatant was stored at -80˚C for further PEEP 0.4 kPa, respiratory rate 40/min, inspiratory oxygen analysis. The remaining 9 BAL-fluid samples were pooled fraction 0.24, TI/E: 1:2. Respiratory settings were adjusted and centrifuged at 1500 rpm for 10 min, the resulting cell- to maintain normocapnia according to hourly performed pellet resuspended and washed twice with PBS. The cells were arterial blood gas analyses. The ventilator was modified for then seeded in polystyrene plates for culture. administration of volatile anaesthetics with a standard vapor (Draeger Medical, Lübeck, Germany). A temperature probe Plasma samples, nitrite assay, ELISA assay. The pooled was inserted rectally in order to keep body temperature blood samples were centrifuged at 5000 rpm for 5 min. Plasma constant at 37-38˚C throughout the experiment. Fluid- filled was collected and stored at -20˚C till further analysis was polyurethane catheters (ID 0.58 mm, OD 0.96 mm, SIMS performed. Portex Ltd., Hythe, UK) were inserted in the right femoral BALF-derived cells were then suspended in RPMI-1640 vein and artery for infusion of anaesthetics and withdrawal of supplemented with 100 U/ml penicillin, 100 μg/ml strepto- blood samples, respectively. mycin, and 10% FCS (Gibco-BRL, Eggenstein, Germany). Cells were seeded on a 24-well polystyrene plate at 0.2x106 Experimental design. Surgical preparation was followed by a cells/well. After initial cultivation for 2 h, non-adherent cells stabilization period of 15 min. Thereafter, animals were were removed. Adherent cells were regarded as alveolar randomly assigned to the study-groups. In the LPS-only macrophages and incubated with 0.5 ml of the aforementioned and Sham-group anaesthesia was maintained by continuous medium. All incubations were performed at 37˚C, 5% CO2 in i.v.
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