0031-3998/01/5004-0515 PEDIATRIC RESEARCH Vol. 50, No. 4, 2001 Copyright © 2001 International Pediatric Research Foundation, Inc. Printed in U.S.A. Prolonged Moderate Hyperoxia Induces Hyperresponsiveness and Airway Inflammation in Newborn Rats DELPHINE DENIS, MICHAEL JOHN FAYON, PATRICK BERGER, MATHIEU MOLIMARD, MANUEL TUNON DE LARA, ETIENNE ROUX, AND ROGER MARTHAN Pediatric Intensive Care and Pulmonology Units, Hôpital Pellegrin-Enfants, Place Amélie Raba Léon, 33076 Bordeaux Cedex, France [D.D., M.J.F.]; and Laboratoire de physiologie cellulaire respiratoire, INSERM E-9937 - Université Victor Segalen Bordeaux 2, 146 rue Léo Saignat, 33076 Bordeaux Cedex, France [D.D., M.J.F., P.B., M.M., M.T.D.L., E.R., R.M.] ABSTRACT Bronchopulmonary dysplasia is the most common cause of neonatal hyperoxic group. Hyperoxia did not influence func- chronic pulmonary disease in premature infants. Airway inflam- tional, morphometric, or cellular data in adult rats. In conclusion, mation appears to play a major pathogenetic role together with exposure of newborn rats to moderate hyperoxia induces airway barotrauma and oxygen toxicity. The aim of the present study hyperresponsiveness and histologic changes similar to those was to determine the effect of a 15-d exposure to moderate reported in bronchopulmonary dysplasia. Hyperresponsiveness hyperoxia (FiO2, 50%) on airway reactivity and inflammatory may be ascribed to an increase in smooth muscle related to the response in neonatal and adult rats. We studied in isolated release of yet undetermined mediators by inflammatory cells tracheal rings the 1) isometric contraction to cumulative concen- infiltrating the airways. Lung immaturity definitely plays a role trations of carbachol (10Ϫ8 to 10Ϫ3 M); 2) epithelial, submuco- because similar alterations are not observed in adult rats. (Pediatr sal, smooth muscle, and connective tissue surface area; and 3) Res 50: 515–519, 2001) distribution of inflammatory cells (mastocytes, granulocytes, macrophages) by using MAb. Reactivity to carbachol was sig- Abbreviations nificantly increased in the hyperoxic pups, in which a 13% BPD, bronchopulmonary dysplasia increase in tracheal smooth muscle surface area was observed. FiO2, fraction of inspired oxygen Type-I mast cells and macrophages (submucosa and connective O2, oxygen tissue) and granulocytes (connective tissue) were increased in the RMCP-I and II, rabbit anti-rat mast cell protease I and II BPD, the most common chronic obstructive pulmonary dis- the etiology of airway hyperreactivity related to BPD (3). ease in children, develops mostly in premature infants who Similar to asthma, pulmonary inflammation is believed to play require prolonged ventilation and/or O2 therapy (1). Airway an important role in the pathogenesis of chronic lung disease. hyperreactivity, which can persist into adolescence, is recog- Histologic features of infants dying from BPD include submu- nized as one of the long-term sequelae of this condition. cosal edema, chronic inflammation, squamous metaplasia of Despite improved neonatal care, including surfactant ther- epithelial cells in large and small airways, thickening of airway apy and new ventilation modalities such as high-frequency smooth muscle, and peribronchiolar fibrosis (4). oscillation, the incidence of BPD is increasing. The precise In the past, various hyperoxic models have been studied. In Ͼ etiology of the disease remains unclear. Barotrauma related to most instances, high FiO2 ( 90%) for short exposure times ventilation induces airway remodeling and hyperreactivity by (Ͻ8 d) has been used (5, 6). Such protocols have induced overdistension of lung tissue (2). However, hyperoxia, which morphologic (airway smooth muscle thickening) as well as causes more significant physiologic, inflammatory, and histo- functional (increased airway hyperresponsiveness) changes in logic changes than barotrauma alone, may play a critical role in young 21-d-old rats (7). However, such protocols did not address the specific issue of lung immaturity because similar Received September 19, 2000; accepted March 1, 2001. effects were observed in the adult rat (7). Moreover, such Correspondence: Michael Fayon, M.D., M.Sc., Service de Réanimation Pédiatrique, conditions of exposure to hyperoxia do not correspond to the Hôpital Pellegrin-Enfants, Place Amélie Raba Léon, 33076 Bordeaux Cedex, France; present-day realities in an intensive care unit. Premature babies e-mail: [email protected] Supported by L’association Bordelaise pour l’avancement des sciences pédiatriques usually benefit from much lower levels of FiO2 for a longer (ABASP). period of time. 515 516 DENIS ET AL. The aim of the present study was to assess the combined period, each preparation was preloaded at the optimal resting ϭ effect of immaturity and realistic (i.e. moderate but prolonged) tension (L0 tension that allows the maximal reproducible hyperoxia on airways. We have established an animal model of active force generation) (11). L0 was determined by generating BPD induced by prolonged but moderate hyperoxia in the a resting tension-force generation curve using acetylcholine newborn rat. We have examined the impact of such exposure (10Ϫ3 M) (Sigma Chemical Co., Saint-Quentin-Fallavier, on airway reactivity, morphology, and inflammation and have France) in both normoxic and hyperoxic pups. L0 corresponded observed that, under these conditions, hyperoxia-induced al- to a passive load of 1750 mg in normoxic pups and 2000 mg terations were restricted to newborn rats. in hyperoxic pups; L0 for adult rats was found to be at 2000 mg in both groups. A cumulative concentration-response curve to Ϫ8 Ϫ3 METHODS carbachol from 10 to 10 M (Sigma Chemical Co., Saint- Quentin-Fallavier, France) was then constructed. For each ring, Exposure to hyperoxia. All animals were housed in our the contractile response to carbachol was expressed both as animal laboratory, which is approved by our Institutional active force (in mg), i.e. the total force minus the resting Board. For each series of experiments, pregnant pathogen-free tension, and as stress (g/mm2), i.e. the active force divided by Wistar rats of known gestational age were purchased. After muscle cross-sectional area. parturition in the laboratory, the pups were redistributed at Emax, which depicts the maximal active force generated random between the mothers. Within 24 h of birth, the pups (final plateau on the cumulative concentration-response curve), were either exposed to moderate hyperoxia (50% O2)ornor- and EC50, the concentration of agonist producing half-maximal moxia (21% O2) at ambient pressure (sea level) for 15 d in response, were calculated for each curve. EC50 was calculated standard cages placed within 86-L capacity Plexiglas isolation by nonlinear curve fitting using a logistic function and ex- chambers (Roller 6 Rothos Sundis, France). This level of pressed as log EC50. Ն exposure, which is in contrast to most previous studies (FiO2 Morphologic study. Subsequent to the organ bath study, 95% for 8 d) (5, 8), was chosen to take into account the change both control tissues and tissues from hyperoxic animals were in the way O2 is now given to neonates in clinical practice (9). embedded in glycolmethacrylate (GMA) (see below) and cut They were housed with one nursing mother during the entire into 2-␮m thick sections perpendicular to the long axis. For the exposure time. morphometric study, sections were stained with toluidin blue. In the hyperoxic group, the O2 fraction in the closed-circuit Light microscopy was performed using an Optiphot micro- chambers was maintained at a constant value of 50 Ϯ 1% by scope (Nikon, Tokyo, Japan). A compartmental analysis was continuous monitoring using an oxygen analyzer (Servomex performed delineating epithelium, conjunctive tissue, submu- ␮ 2 O2 Analyzer 580A, England) that controlled an electric valve cosa, and smooth muscle layer. The area ( m ) and epithelium ␮ at the output of the O2 tank. Gas circulation was facilitated by thickness ( m) (layer area divided by the length of the epithe- an air pump (3.5 L/min). In the control group, rats were housed lial basement membrane) were calculated using a video inter- in a similar chamber, and ambient air was continuously flushed active display system (ϫ40 magnification) and an appropriate through the control chambers via an identical air pump. Moth- software (Quancoul, Quant’Image 1995-7, France) as previ- ers were given food and water ad libitum. Cages were removed ously described (12). Mean tracheal smooth muscle cross- from the isolation chambers for 5 min every day to ensure sectional area (n) was determined from the data obtained in servicing. Moisture and CO2 were absorbed with silica gel and three nonadjacent sections of the same trachea performed soda lime (Prolabo, France), respectively, and both were perpendicularly to the long axis of the specimen. changed every day. Pairs of adult rats were also exposed either Sample processing for immunohistochemistry. The tracheal to hyperoxia or normoxia for 15 d under the same conditions as specimens embedded in GMA were also used for immuno- rat pups. staining as described previously (12, 13). The GMA sections Isometric contraction measurement. On d 15, the animals were cut at 2-␮m thickness perpendicular to the long axis with were removed from their chambers and anesthetized by intra- an ultramicrotome and incubated overnight at room tempera- peritoneal administration of ethyl carbamate (pups, 80 mg; ture with mouse or rabbit antibodies (Ab) including