Pediatric Pulmonology 41:55–60 (2006)
Dexamethasone Alleviates Meconium-Induced Airway Hyperresponsiveness and Lung Inflammation in Rabbits Juraj Mokry, MD, PhD,1* Daniela Mokra, MD, PhD,2 Martina Antosova, Mgr,1 Janka Bulikova,2 Andrea Calkovska, MD, PhD,2 and Gabriela Nosalova, MD, DSc1 Summary. The effects of dexamethasone on in vitro airway reactivity associated with lung inflammation were investigated in rabbits with meconium aspiration. Oxygen-ventilated adult rabbits received an intratracheal bolus of 4 ml/kg body weight of saline (Sal, n ¼ 4) or human meconium (25 mg/ml). Thirty minutes later, meconium-instilled animals intravenously received 0.5 mg/kg of dexamethasone (Dexa, n ¼ 6), or were left without treatment (Meco, n ¼ 5). The animals were ventilated for a further 5 hr and then sacrificed. The left lungs were lavaged with saline, and the white blood cell (WBC) count was estimated. Tracheal and right-lung tissue strips were placed into organ chambers with Krebs-Henseleit solution. Cumulative doses of histamine (108 –103 mol/l) and acetylcholine (108 –103 mol/l) were added to the chambers, and recordings of contractions were made after a 30-min loading phase with a tension of 4 grams, and another 30-min adaptation phase with a tension of 2 g. Tracheal smooth muscle in vitro reactivity to histamine was higher in the Meco than in the Sal group, and dexamethasone decreased the reactivity compared to the Meco group (P < 0.05). Lung tissue in vitro reactivity to histamine was slightly higher in the Meco than in the Sal group (P > 0.05), and dexamethasone decreased the reactivity compared to both the Meco and Sal groups (P < 0.05). No between-group differences were observed in tracheal or lung in vitro reactivity to acetylcholine (P > 0.05). In the Meco group, blood WBC (P > 0.05) and neutrophil (P < 0.05) counts were lower than in the Sal and Dexa groups. Lung neutrophils and eosinophils were higher in both the Meco and Dexa groups than in the Sal group (P < 0.01). Dexamethasone decreased neutrophils (P < 0.05) compared to the Meco group. Meconium-induced airway hyperreactivity to histamine and lung inflammation were alleviated by dexamethasone. Pediatr Pulmonol. 2006; 41:55–60. ß 2005 Wiley-Liss, Inc. Key words: airway reactivity; dexamethasone; inflammation; lung; meconium; rabbit.
INTRODUCTION
Meconium aspiration syndrome (MAS) is the major cause of respiratory failure in term newborns. Airway obstruction, inactivation of pulmonary surfactant, lung inflammation, and vasoconstriction due to meconium aspiration are responsible not only for acute respiratory failure, but also for increased respiratory morbidity during infancy.1 Proinflammatory substances present in meconium,2 as well as cytokines produced by activated leukocytes and lung cells, impair pulmonary epithelium, surfactant, and vasoreactivity. The hyperresponsiveness of pulmonary vascular smooth muscle in MAS was previously shown,3 but there is a lack of information regarding the influence of meconium aspiration on airway reactivity. Recently, meconium instillation in a murine model increased in vivo airway responsiveness to methacholine, associated with lymphocytic/eosinophilic inflammation and elevated concentrations of interleukins in bronchoalveolar lavage fluid.4 A higher prevalence of asthmatic symptoms and abnormal bronchial reactivity in later childhood in infants who suffered in the neonatal period ß 2005 Wiley-Liss, Inc.
from MAS may also support the supposititon of higher airway reactivity due to meconium aspiration.5 1 Department of Pharmacology, Jessenius Faculty of Medicine, Comenius University, Martin, Slovakia. 2
Department of Physiology, Jessenius Faculty of Medicine, Comenius University, Martin, Slovakia. This paper was presented in part at the 81st Czech and Slovak Physiological Days, February 2–4, 2005, Kosice, Slovakia. Grant sponsor: Agency for Science and Ministry of Education; Grant number: 1/2306/05; Grant sponsor: Comenius University; Grant number: 43/2004. *Correspondence to: Juraj Mokry, Department of Pharmacology, Jessenius Faculty of Medicine, Comenius University, Sklabinska 26, 03753 Martin, Slovakia. E-mail:
[email protected] Received 25 July 2005; Accepted 3 August 2005. DOI 10.1002/ppul.20330 Published online 14 October 2005 in Wiley InterScience (www.interscience.wiley.com).
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Nowadays, many therapeutic approaches are being tested in the management of MAS. Recently, we combined exogenous surfactant administration and high-frequency ventilation.6,7 Also seemingly of promise is the coadministration of exogenous surfactant and nonionic polymers,8 inhaled nitric oxide,9 or liquid ventilation.10 However, these approaches do not directly influence the inflammation process generated in lungs after meconium aspiration. Regarding these facts, the administration of corticosteroids in severe MAS may be of benefit. Although the previous experimental administration of hydrocortisone did not improve lung function,11,12 in recent studies, methylprednisolone and dexamethasone reduced lung vascular resistance and pulmonary artery pressure in piglets,13–15 and improved lung function in newborns with MAS.16 These experiments were performed to investigate changes of in vitro airway reactivity associated with inflammatory changes in the lungs of rabbits after meconium instillation. Furthermore, we evaluated the effects of intravenously administered dexamethasone on both in vitro airway reactivity and lung inflammation in rabbits with MAS after a 5.5-hr artificial ventilation with 100% oxygen. MATERIALS AND METHODS Meconium
Meconium was collected from 20 healthy term neonates. Samples were pooled, lyophilized, and stored at 208C. Before use, meconium was suspended in 0.9% NaCl at a concentration of 25 mg/ml. Design of Experiments
The study design was approved by the Local Ethics Committee of the Jessenius Faculty of Medicine. Adult rabbits (chinchilla) with a mean body weight (b.w.) of 2.0 0.3 kg were anesthetized with intramuscular ketamine (Narkamon, Spofa, Czech Republic) at a dose of 20 mg/kg b.w. and xylazine (Rometar, Spofa, Czech Republic) at a dose of 5 mg/kg b.w., followed by intravenous ketamine at a dose of 20 mg/kg/hr. Tracheotomy was performed, and a tracheal tube was inserted. The femoral artery was cannulated for sampling arterial blood, the femoral vein for administration of drugs and anesthetics, and the jugular vein for sampling mixed venous blood. Animals were paralyzed with pipecuronium bromide (Arduan, Gedeon Richter, Hungary) at a dose of 0.3 mg/kg b.w./30 min intravenously (i.v.) to avoid spontaneous breathing, and were ventilated with the pressure-controlled ventilator Beat-2 (Chirana, Slovakia), with a frequency of 30/min, fraction of inspired oxygen (FiO2) of 0.21, positive end-expiratory pressure (PEEP) of 0 kPa, and inspiration time (Ti) of 50%. Baseline blood samples were taken. Animals were instilled with saline (Sal, n ¼ 4) or a suspension of meconium at a dose of
4 ml/kg b.w. proportionally into the left and right lungs during the lateral positioning of the animal. From this moment, animals were ventilated with 100% oxygen and a PEEP of 0.3 kPa. Within 30 min after meconium instillation, respiratory failure developed, defined as a >30% decrease in dynamic lung-thorax compliance and partial pressure of oxygen in arterial blood (PaO2) 0.05). Dexamethasone significantly decreased lung smooth muscle reactivity to histamine compared to the Meco group at histamine concentrations of 106 –104 mol/l (P < 0.05), and comapred to the Sal group at histamine concentrations of 105 –104 mol/l (P < 0.05, Fig. 2). 400
Contraction (mN)
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Fig. 2. Contractile responses of lung smooth muscle after cumulative doses of histamine. Between-group comparisons, Meco vs. Sal at all concentrations, P > 0.05; Dexa vs. Meco at concentrations of 106 –104 mol/l, P < 0.05; Dexa vs. Sal at concentrations of 105 –104 mol/l, P < 0.05.
No significant between-group differences were observed in tracheal or lung smooth muscle reactivity to acetylcholine (P > 0.05, data not shown). WBC Count in Peripheral Blood
Blood leukocytes slightly decreased after both saline and meconium administration, but without significant differences between groups (P > 0.05). The total WBC count then increased in all groups, with the lowest value in the Meco group at the end of the experiment (2.8 0.3 109/l in the Meco group vs. 3.9 0.3 109/l in the Sal group, and vs. 3.4 0.7 109/l in the Dexa group). However, the differences between groups were not significant (P > 0.05). At the end of the experiment, in all groups, the neutrophil count was higher when compared to values before meconium instillation (P < 0.001), however, neutrophils were significantly lower in the Meco group than in the Sal and Dexa groups (P < 0.05, Fig. 3). In addition, in the Meco group, the eosinophil count at the end of the experiment was significantly higher when compared to the value before meconium instillation (P < 0.05, Fig. 4). However, no significant differences between groups were found in the eosinophil count (P > 0.05).
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WBC Count in Lavage Fluid Sediment 0 -8
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Histamine (log M)
Fig. 1. Contractile responses of tracheal smooth muscle after cumulative doses of histamine. Between-group comparisons, Meco vs. Sal at concentration of 108 and 107 mol/l, P < 0.05; Dexa vs. Meco at concentrations of 108 –104 mol/l, P < 0.05; and Dexa vs. Sal at all concentrations, P > 0.05.
In the total WBC count, the differences between groups were uncountable under the microscope. In the Meco and Dexa groups, increased neutrophils and eosinophils were observed compared to the Sal group (P < 0.01). However, in the Dexa group, significantly lower neutrophil (P < 0.05) and nonsignificantly lower eosinophil counts were found compared to the Meco group (P > 0.05; Fig. 5).
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Blood neutrophil count (%)
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0 Before M
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Fig. 3. Blood neutrophil count (%) during experiments. Before M, before meconium administration. Between-group comparisons, Meco vs. Sal at 5 hr, P < 0.05; Dexa vs. Meco at 5 hr, P < 0.05. Within-group comparisons: in all groups, 5 hr vs. Before M, P < 0.001.
DISCUSSION
Meconium instillation deteriorates lung compliance and oxygenation within several minutes.7 Ongoing tissue hypoxia combined with hyperoxia from oxygen therapy, together with the action of proinflammatory substances, leads in a short time to the injury of surfactant and alveolocapillary membrane. Flooding the lungs with edema fluid and a migration of polymorphonuclear neutrophils from the blood further complicate the situation. In this study, meconium administration significantly increased the total number of WBC (particularly neutrophils in the lungs) compared to the saline group. The higher count of neutrophils in the lungs was linked with their decrease in peripheral blood. Comparably, in pigs with meconium
Blood eosinophil count (%)
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Sal Meco Dexa
0 Before M
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Fig. 4. Blood eosinophil count (%) during experiments. Before M, before meconium administration. Between-group comparisons, all P > 0.05. Within-group comparison, Meco group, 5 hr vs. Before M, P < 0.05.
aspiration,13 as well as in other lung diseases, the loss of circulating neutrophils and the extent of their lung accumulation were proportional to the magnitude of lung injury.19 The airway narrowing in MAS is caused not only by mechanical obstruction by aspirated meconium, but also by peribronchial edema due to the action of various inflammatory mediators. The release of these mediators, including arachidonic acid metabolic products from activated cells during inflammation, modulates the smooth muscle tone and its responses to contractile mediators,20,21 and may thus contribute to airway hyperreactivity. Whereas airway inflammation is usually associated with increased in vitro smooth muscle reactivity,17,22 we tested in vitro airway reactivity in rabbits with meconium aspiration. Meconium instillation increased airway reactivity to histamine, confirming the importance of inflammation and bronchoconstriction in the pathogenesis of MAS. However, by this method it is not possible to distinguish which kind of smooth muscle is predominantly responsible for lung tissue hyperresponsiveness, since the lung tissue strips contained both airway and vascular smooth muscle parts. However, significantly increased reactivity of tracheal smooth muscle strips indicates that the action of airway smooth muscle could be superior in this situation. Costello et al., in ovalbumine-senzitized guinea pigs, found that hyperreactivity to histamine is vagally mediated and dependent on eosinophil recruitment.23 An association between in vivo airway hyperresponsiveness and eosinophilic inflammation after meconium aspiration in a murine model was also described by Khan et al.4 We are aware that the adult rabbit model does not fully mimic the situation in developing lungs of the newborn. In this study, we avoided the influence of transitional structural and functional changes by studying adapted rabbit lungs. Our results showed that meconium aspiration increases airway reactivity in adult rabbits with MAS. However, further experiments using neonatal animals are needed to elucidate to what extent the role of increased airway reactivity is significant in the pathogenesis of neonatal MAS. Due to only localized lung inflammation after meconium instillation, Korhonen et al. concluded that local treatments such as surfactant lung lavage might be more beneficial in MAS than systemic anti-inflammatory treatment.24 However, several studies showed a benefit of corticosteroids in MAS.13,15,16 Corticosteroids reduce edema formation through diminished endothelial damage and microvascular permeability.13 In addition, they were suggested to inhibit the phospholipase A2 (PLA2) synthesis induced by meconium.15 However, corticosteroids affect many other steps in the inflammatory response in MAS. Administration of dexamethasone recently inhibited the meconium-stimulated expression of
Dexamethasone and Airway Reactivity in MAS
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Fig. 5. Neutrophil and eosinophil counts in lavage fluid (%). Between-group comparisons: neutrophils, Meco vs. Sal, P < 0.001; Dexa vs. Meco, P < 0.05; Dexa vs. Sal, P < 0.001. Eosinophils, Meco vs. Sal, P < 0.001; Dexa vs. Meco, P > 0.05; Dexa vs. Sal, P < 0.01.
cyclooxygenase-2 and iNO-synthase in respiratory epithelium, macrophages, and endothelial cells.25,26 Interruption of the cytokine cascade and inhibition of chemotactic factors by corticosteroids may result in a reduction of neutrophil margination and influx into the lungs, with a simultaneous increase of the blood leukocyte count.13,15 Similarly, in this study, dexamethasone decreased neutrophils in the lungs, and increased circulating neutrophils. In addition, dexamethasone reduced in vitro meconiuminduced hyperreactivity of tracheal and lung tissue strips to histamine, and diminished counts of both circulating and lung eosinophils. This corresponds with the results of Wolley et al., who showed that airway reactivity, as well as the activity of airway eosinophils in dogs, decreased after administration of corticosteroids.27 Yost et al. observed decreased airway responsiveness after dexamethasone in antigen-challenged guinea pigs due to increased cholinesterase activity and inhibitory M2 receptor function.28 Similarly, Evans et al. showed that dexamethasone prevents the recruitment of eosinophils to the airways and to airway nerves, resulting in decreased antigeninduced hyperreactivity by protecting neuronal M2muscarinic receptors from antagonism by the eosinophil major basic protein.29 Parasympathetic control of airway caliber is mediated through the release of acetylcholine binding to muscarinic receptors (especially the M3 subtype) on airway smooth muscle.30 Therefore, we evaluated the in vitro effects of cumulative doses of acetylcholine on the contractile responses of tracheal and lung smooth muscle under conditions of meconium instillation, compared to saline instillation and treatment with dexamethasone. However, we observed no significant differences between groups. This finding could be explained by the stimulation of other muscarinic receptor subtypes, especially the neuronal presynaptic M2 receptor subtype, whose normal function or dysfunction is asso-
ciated with asthma and other inflammatory diseases.29 Furthermore, dexamethasone does not inhibit vagally induced bronchoconstriction via a decrease in airway reactivity at the level of M3 muscarinic receptors.29 The ability of intravenous acetylcholine to cause bronchoconstriction in vagotomized guinea pigs is unaltered by antigen challenge, as also confirmed by these in vitro experiments. In this study, meconium instillation in rabbits caused significant lung inflammation within several hours, and increased in vitro airway smooth muscle reactivity to histamine. Polymorphonuclear accummulation in the lungs and increased in vitro airway reactivity were alleviated by the administration of dexamethasone, pointing to its possible use in the treatment of severe meconium aspiration syndrome. Nevertheless, it is difficult to distinguish whether the observed reduction of meconiuminduced airway hyperreactivity by dexamethasone was predominantly caused by reduced polymorphonuclear migration, by the release of inflammatory mediators and edema, or by a direct bronchodilating effect. Therefore, testing anti-inflammatory and/or bronchodilator drugs other than corticosteroids in the future could bring more light to this problem. ACKNOWLEDGMENTS
We thank M. Duchonova, D. Kuliskova, P. Kuzma, M. Repcakova, and S. Svorkova for technical assistance. REFERENCES 1. Yuksel B, Greenough A, Gamsu HR. Neonatal meconium aspiration syndrome and respiratory morbidity during infancy. Pediatr Pulmonol 1993;16:358–361. 2. de Beaufort AJ, Bakker AC, van Tol MJD, Poorthuis BJ, Schrama AJ, Berger HM. Meconium is a source of pro-inflammatory substances and can induce cytokine production in cultured A549 epithelial cells. Pediatr Res 2003;54:491–495.
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